Peptide Analogues of PA-IL and Their Utility for Glycan and Glycoconjugate Analysis and Purification

ABSTRACT

Provided are peptide analogues of PA-IL and compositions containing them. The PA-IL peptide analogues have altered carbohydrate binding specificity relative to a PA-IL of SEQ ID NO:1, and thus the analogues contain amino acid substitutions in SEQ ID NO:1. The substitutions can be at positions 50, 52 and 53 of SEQ ID NO:1 and can include combinations of amino acid substitutions at those positions 
     Also included are methods for detecting changes in the glycosylation of carbohydrates and for separating biomolecules which contain glycoproteins or glycoconjugates.

INTRODUCTION

Glycosylation is one of the most abundant and biologically significantpost-translational modifications to occur in cells. It is a highlycomplex non-template driven process that results in the addition ofoligosaccharide moieties to a variety of biomolecules. Cell surfaces,both prokaryotic and eukaryotic, are covered in a dense layer of complexoligosaccharide structures that are attached to proteins and lipids.This layer is called the “glycocalyx” and the nature of the glycansdisplayed can be organism and cell type specific. As the interfacebetween a cell and its environment it is not surprising thatinteractions between these glycans with carbohydrate binding proteins,called lectins, mediate a vast array of biological processes, play acentral role in the orchestration of the immune system and mediateinteractions between cells and various infectious agents such as prions,viruses and microorganisms. The glycans present on proteins also have avery significant impact on their physiochemical properties andbiological activity. Changes in the glycans presented on glycoproteinsor cell surfaces can result in, or be indicative of, changes in thephysiological status of a cell or signify the development of a diseasestate (1-6) including many types of cancer (7-10) and autoimmunedisorders such as rheumatoid arthritis (11-14). Many biopharmaceuticalmolecules are also glycosylated proteins and the glycans attached tothese products can have a significant impact on their safety andefficacy. Given the biological significance of glycosylation, there is arequirement for methods that enable efficient isolation of glycosylatedbiomolecules and informative glycoanalysis of biomolecules and cellsurfaces.

Lectins are carbohydrate proteins that are capable of recognizing andbinding reversibly to specific carbohydrate structures. They displayexquisite specificity for their cognate glycans and their ability todiscriminate between different glycan structures has been exploited formany years for glycoanalytical applications. Their ability to bind toglycans in situ on proteins and cell surfaces, without the need forprior release and derivatization, makes them particularly attractivewhen compared to alternative MS and HPLC based approaches as thesetreatments can often result in the loss of significant biological data.When immobilized to solid support matrices, lectins can be used toeffect the separation and purification of glycosylated molecules. Lectinaffinity chromatography is often used as a preliminary step to isolateor separate oligosaccharides, glycopeptides, glycoproteins andglycoprotein glycoforms to facilitate their identification andcharacterisation.

The most commonly used lectins are plant lectins and these havetraditionally exhibited significant problems, particularly with respectto product quality and performance. Many plant lectins are purified fromsource material, due to incompatibility with recombinant productionmethods, and this results in batch to batch variations and variabilityfrom one supplier to another (5,15,16). Production methods usuallygenerate relatively low yields and final products are expensive whichhas meant that lectins have been restricted to analytical scaleapplications where only small quantities are required (16).

Prokaryotic lectins offer new opportunities for the development ofsuperior glycoselective bioaffinity tools but, to date, they have beenrelatively underexploited. They usually exhibit greater affinities fortheir glycan targets and less structural complexity than plant lectins(17). They are also more amenable to recombinant production,particularly in Escherichia coli, which simplifies production but alsoopens up opportunities for the development of novel enhanced recombinantprokaryotic lectins (RPL's) with diversified and optimized bindingproperties (18-20).

We will demonstrate herein how the carbohydrate binding properties ofthe α-galactophilic PA-IL protein, from the opportunistic pathogenPseudomonas aeruginosa (21-23), were significantly altered throughrandom mutagenesis of specific amino acid residues in the proteinscarbohydrate binding site. We will generate a series of novel RPL's thatexhibit specificity and high affinity for glycoprotein targetsdisplaying lactosamine (LacNAc) and demonstrate that binding wasdependent on terminal β1,4-linked galactose. Lactosamine is commonlydisplayed as part of glycan structures found on cell surfaces and aspart of the antenna of N-linked glycans displayed on glycoproteinsincluding serum IgG's where it is important for the ability of thesemolecules to elicit CDC (complement dependent cytotoxicity) and ADCC(antibody dependent cellular cytotoxicity) effector functions(11,12,14,24). RPL's with specificity for LacNAc therefore representpotentially valuable tools for glycoselective applications throughoutthe life sciences.

These novel RPL's carried multiple simultaneous substitutions in thecarbohydrate binding site of the PA-IL (Pseudomonas aeruginosa lectin 1or Pseudomonas aeruginosa lectin I) protein. As a result, it wasdifficult to fully determine the specific contribution of individualsubstitutions to the observed carbohydrate binding properties of themutant PA-IL proteins. In this work, we also undertook a progressivesite directed mutagenesis approach to assess the significance ofspecific amino acid residues in dictating binding specificity andaffinity and, through in silico modelling, we explored the potentialstructural basis for the observed carbohydrate binding properties. Indoing so, we identified optimal amino acid substitutions that promotespecific carbohydrate binding activities and produced an array of novelRPL's with diverse carbohydrate binding activities that will be of usefor a broad spectrum of glycoselective applications.

STATEMENTS OF INVENTION

In a first embodiment, there is provided a peptide analogue of PA-IL ofSEQ ID NO: 1, wherein the peptide analogue has altered carbohydratebinding specificity, and wherein the peptide analogue comprises an aminoacid substitution at one, two or three of positions 50, 52 and 53,wherein the amino acid substitution at position 50 is selected from thegroup consisting of Ala, Val, Leu, Ile, Met, Phe, Trp, Pro, Ser, Thr,Cys, Tyr, Gly, Asn, Asp, Gln, Glu, Lys, and Arg; optionally from thegroup consisting of Ala, Val, Leu, Phe, Pro, Ser, Thr, Gly, Asn, Asp,Gln, Glu, Lys, and Arg; and further optionally from the group consistingof Ala, Val, Leu, Ser, Thr, Gly, Asn, Gln, Glu, Lys and Arg; wherein theamino acid substitution at position 52 is selected from the groupconsisting of Ser, Thr, Cys, Asn, Gln, Asp, Glu, Lys, Arg and His;optionally from the group consisting of Thr, Cys, Asn, Arg and His; andfurther optionally from the group consisting of Asn, Thr, Arg and His;and wherein the amino acid substitution at position 53 is selected fromthe group consisting of Ala, Val, Leu, Ile, Met, Phe, Trp, Pro, Gly,Ser, Thr, Cys, Tyr, Asn, Asp, Glu, Lys, Arg and His; optionally from thegroup consisting of Ala, Val, Leu, Gly, Ser, Tyr, Asn, Asp, Glu, Lys,Arg and His; and further optionally from the group consisting of Ala,Val, Leu, Gly, Ser, Asn, Asp, Glu, Lys, Arg and His.

Optionally, the peptide analogue has improved binding to a carbohydratehaving a terminal 13 galactose, optionally a terminal 131,4-linkedgalactose, over PA-IL of SEQ ID NO: 1 and wherein the peptide analoguecomprises an amino acid substitution at position 50 selected from Ala,Val, Leu, Ile, Met, Pro, Ser, Thr, Cys, Asn, Gln, Glu, Lys and Arg;optionally from the group consisting of Ala, Val, Leu, Pro, Ser, Thr,Asn, Gln, Glu, and Lys; and further optionally from the group consistingof Asn, Gln, Glu, and Val. Further optionally, the peptide analoguecomprises Asn at position 50 and an amino acid substitution at position53 selected from the group consisting of Ala, Val, Leu, Ile, Met, Gly,Ser, Thr, Asn, Asp, Glu, Lys, Arg and His; optionally from the groupconsisting of Ala, Val, Leu, Gly, Ser, Asn, Asp, Glu, Lys, Arg and His;and further optionally from the group consisting of Ala, Val, Gly, Ser,Lys, Arg and His. Alternatively, the peptide analogue comprises Asn atposition 50 and Gly at position 53; the peptide analogue optionallycomprising Gln, Asp, Glu or Asn at position 52; the peptide analoguefurther optionally comprising Asn at position 52. Further alternatively,the peptide analogue comprises Val at position 50 and an amino acidsubstitution at position 53 selected from the group consisting of Ala,Val, Leu, Ile, Met, Gly, Ser, Thr, Asn, Asp, Glu, Lys, Arg and His;optionally from the group consisting of Ala, Val, Leu, Gly, Ser, Asn,Asp, Glu, Lys, Arg and His; and further optionally from the groupconsisting of Ala, Val, Gly, Ser, Lys, Arg and His.

Optionally, the peptide analogue has altered carbohydrate bindingspecificity for a carbohydrate having a terminal α-linked galactose overPA-IL of SEQ ID NO: 1 and wherein the peptide analogue comprises anamino acid substitution at position 50 selected from Ala, Val, Leu, Ile,Met, Ser, Thr, Cys, Asn, Gln, Glu, Lys and Arg; optionally from thegroup consisting of Ala, Val, Leu, Ser, Thr, Asn, Gln, Glu, Lys and Arg;and further optionally from the group consisting of Val, Leu, Asn, Glnand Lys. Further optionally, the peptide analogue comprises Asn atposition 50 and an amino acid substitution at position 53 selected fromthe group consisting of Ala, Val, Leu, Ile, Met, Gly, Ser, Thr, Cys,Asn, Asp, Glu, Arg, Lys and His; optionally from the group consisting ofAla, Val, Leu, Gly, Ser, Asn, Asp, Glu, Arg, Lys and His; and furtheroptionally from the group consisting of Ala, Ser, Gly, Arg, Lys and His.

Optionally, the peptide analogue has enhanced carbohydrate bindingspecificity for a carbohydrate having a terminal α-linked galactose overPA-IL of SEQ ID NO: 1 and wherein the amino acid substitution atposition 53 is selected from the group consisting of Asn, Asp, Glu, Argand His; and optionally from the group consisting of Glu and Arg.

Optionally, the peptide analogue has altered carbohydrate bindingspecificity for a carbohydrate having a terminal α-linked galactose overPA-IL of SEQ ID NO: 1 and wherein the peptide analogue comprises Val atposition 50 and an amino acid substitution at position 53 selected fromthe group consisting of Ala, Val, Leu, Ile, Met, Phe, Trp, Pro, Gly,Ser, Thr, Cys, Tyr, Asn, Asp, Glu, Lys, Arg and His; wherein optionallythe peptide analogue comprises Val at position 50 and an amino acidsubstitution at position 53 selected from the group consisting of Ala,Val, Leu, Gly, Ser, Tyr, Asn, Asp, Glu, Lys, Arg and His and furtheroptionally, the peptide analogue comprises Val at position 50 and anamino acid substitution at position 53 selected from the groupconsisting of Arg and Lys.

Optionally, the peptide analogue has altered carbohydrate bindingspecificity for a carbohydrate having a terminal α-linked galactose overPA-IL of SEQ ID NO: 1 and wherein the peptide analogue comprises Gln atposition 50 and an amino acid substitution at position 53 selected fromthe group consisting of Ala, Val, Leu, Ile, Met, Phe, Trp, Pro, Gly,Ser, Thr, Cys, Tyr, Asn, Asp, Glu, Lys, Arg and His; wherein optionallythe peptide analogue comprises Gln at position 50 and an amino acidsubstitution at position 53 selected from the group consisting of Ala,Val, Leu, Gly, Ser, Tyr, Asn, Asp, Glu, Lys, Arg and His and furtheroptionally, the peptide analogue comprises Gln at position 50 and anamino acid substitution at position 53 selected from the groupconsisting of Arg and Lys.

Optionally, the peptide analogue has enhanced carbohydrate bindingspecificity for a carbohydrate having a terminal β- or a α-linkedgalactose over PA-IL of SEQ ID NO: 1 and wherein the peptide analoguecomprises an amino acid substitution at position 50 selected from Asn,Leu and Gln.

Optionally, the peptide analogue comprises an amino acid substitution atposition 50 selected from Ala, Val, Leu, Phe, Pro, Gly, Ser, Thr, Asn,Gln, Asp, Glu, Lys, and Arg.

Optionally, the peptide analogue comprises Asn at position 50 and anamino acid substitution at position 53 is selected from the groupconsisting of Ala, Val, Leu, Gly, Ser, Tyr, Asn, Asp, Glu, Lys, Arg andHis.

Optionally, the peptide analogue comprises an amino acid substitution atposition 53 selected from Glu, Lys and Arg.

Optionally, the peptide analogue comprises Val at position 50 and anamino acid substitution at position 53 selected from the groupconsisting of Ala, Val, Leu, Gly, Ser, Tyr, Asn, Asp, Glu, Lys, Arg andHis.

Optionally, the peptide analogue comprises an amino acid substitution atposition 52 selected from the group consisting His, Asn, Cys, Thr andArg and, further optionally, an amino acid substitution at position 50selected from the group consisting of Leu, Thr, Val, Asn, Gly and Proand an amino acid substitution at position 53 selected from the groupconsisting of Arg, Glu, Ser, Gly, Leu and Asn.

Optionally, the carbohydrate, for which the peptide analogue of thepresent invention has altered carbohydrate binding specificity, is acarbohydrate or is selected from the group consisting of glycoprotein,glycoconjugate and cell surface. Further optionally, the glycoprotein,glycoconjugate and cell surface comprises an oligosaccharide or apolysaccharide linked to a protein or other conjugate.

In a second embodiment, there is provided a method for detecting changesin the glycosylation of a carbohydrate that is optionally selected fromthe group consisting of glycoprotein, glycoconjugate and cell surface,the method comprising qualitatively or quantitatively assessing terminalgalactosylation using a peptide analogue of any aspect of the firstembodiment. These methods find utility for detecting changes in thepresence of, or exposure of, terminal α- and β-linked galactose, whetherfor purification or analytical work, or for diagnostic purposes.

In a third embodiment, there is provided a method of separating andisolating/purifying biomolecules/cells comprising a glycoprotein orglycoconjugate, the method comprising contacting the peptide analogue ofany aspect of the first embodiment with a solution or suspensioncontaining biomolecules/cells; separating any biomolecules/cells notbound by the peptide analogue and, optionally, subsequently recoveringbiomolecules/cells bound by the peptide analogue by disassociating themfrom the peptide analogue.

LEGENDS TO FIGURES

FIG. 1: Structure of the PA-IL Protein. (A) Tetrameric PA-IL proteinwith bound iGb3 trisaccharide Gal-α1,3-Gal-β1,3-Glc (PDB code 2VXJ)(23). Each monomer subunit (A, B, C, D) contains a single carbohydratebinding site. A single calcium ion is coordinated within each bindingsite (grey sphere) and is essential for sugar binding. (B) The PA-ILbinding site showing coordination of the calcium ion and binding ofiGb3. Amino acid residues Asp100, Thr104, Asn107, Asn108 of the calciumbinding loop (residues 100-108), and Tyr36 of the neighbouring loop, areinvolved in calcium coordination and also form hydrogen bonds(interactions indicated by dash lines) with the non-reducing α-linkedgalactose. The non-reducing α-linked galactose also participates incoordination of the calcium contributing two interactions with the metalion. Residues His50 and Gln53 form hydrogen bonds with the non-reducingaGal and Gln53 also interacts with the second galactose in thetrisaccharide. Images were generated using Deep View (Swiss Model) (25)and rendered using CCP4MG software (26).

FIG. 2: Qualitative ELLA Screening of Random rPA-ILNm Proteins. NinerPA-ILNm proteins, identified in initial library screens, were purifiedand re-assessed for their ability to bind to (A) transferrin and derivedtransferrin glycoforms and (B) fetuin and derived fetuin glycoforms. Thedata confirmed the ability of these rPA-ILNm proteins to bind toglycoprotein targets displaying LacNAc (asialotransferrin andasialofetuin) and that binding was dependent on the terminal β1,4-linkedgalactose (note the loss of signals on agalactotransferrin andagalactofetuin).

FIG. 3: Identification of Amino Acid Substitutions Present in IsolatedrPA-ILNm Proteins. Sequence alignment of amino acid residues from Gln45to His59 in PA-IL with the equivalent residues present in isolatedrPA-ILNm proteins. Specific amino acid substitutions were observed tooccur with high frequency. Substitutions occurring more than once ateach of the positions corresponding to HSO, D52 and Q53 in wild typenative PA-IL protein are indicated in boxes.

FIG. 4: Lectin Dilution Response Curves against Defined BSAGlycoconjugates. (A) BSA-LacNAc: All of the rPA-ILNm proteins, with theexception of rPA-ILNmA8, displayed strong binding to BSA-LacNAc but therPA-ILNmE6 exhibited the highest relative affinity to thisglycoconjugate, with signal reaching saturation at lectin concentrationsabove 0.625 μg mL⁻¹. As expected, the parental rPA-ILN protein showedlittle binding to this glycoconjugate, even at relatively high lectinconcentrations of 10 μg L⁻¹. (B) BSA-αGal: The parental rPA-ILN proteinexhibited the strongest binding to the BSA-αGal glycoconjugate, withsignals reaching saturation at lectin concentrations above 1.25 μg mL⁻¹.Four rPA-ILNm proteins were also observed to bind significantly to thisglycoconjugate. The rPA-ILNmB10 and rPA-ILNmF3 proteins displayedsimilar response curves and a higher relative affinity for thisglycoconjugate than rPA-ILNmB4. The rPA-ILNmE6 was also observed to bindcomparatively weakly to BSA-αGal and signals did not reach saturationlevels over the lectin concentration range examined. (C) GlycanSelectivity: indicated by plotting the ELLA response of each lectin toeach glycoconjugate at a lectin concentration of 0.625 μg mL⁻¹. Thisclearly shows that the rPA-ILNmE6 protein exhibits significantpreferential binding to BSA-LacNAc while rPA-ILNmF3 appears to exhibit adual binding specificity for both glycoconjugates.

FIG. 5: Evaluation of rPA-ILNm Binding to Natural Glycoprotein Targets.Lectin dilution response curves were prepared against (A)asialotransferrin and (B) asialofetuin. Biotinylated ECL was included asa positive control in ELLA's. The rPA-ILNmE6 displayed the highestrelative affinity to both glycoproteins with signals reaching saturationlevels on both glycoproteins at lectin concentrations above 1 μg mL⁻¹.The rPA-ILNmC5, rPA-ILNmG3 and rPA-ILNmB4 exhibited strong binding toasialofetuin but displayed significantly reduced relative affinities toasialotransferrin. This indicated an overall lower relative affinity forLacNAc and a greater dependency on glycan display density than therPA-ILNmE6 protein. ECL displayed a high relative affinity to bothglycoprotein targets but responses on asialotransferrin indicate anoverall lower relative affinity for this glycoprotein compared torPA-ILNmE6.

FIG. 6: Determination of Affinity Constants for rPA-ILNm Proteinsagainst BSA-LacNAc. Glycoconjugate dilution response curves wereprepared and used to determine the B_(max) and the affinity constantK_(D) for each of the rPA-ILNm proteins according to the methoddescribed by Kirkeby et al 2002 (27). ECL was also included forcomparative purposes. Both rPA-ILNmE6 and rPA-ILNmF3 displayed higherrelative affinities for BSA-LacNAc than ECL as indicated by their lowercalculated K_(D) values. The rPA-ILNmE6 and rPA-ILNmF3 proteinsgenerated K_(D) values of 4 ng and 6.3 ng respectively while ECLgenerated a K_(D) value of 21 ng. This indicates that the rPA-ILNmE6 hasa five fold higher relative affinity for BSA-LacNAc than ECL while therPA-ILNmF3 has approximately a 3 fold higher relative affinity.

FIG. 7: The Application of rPA-ILNmE6 for the Separation and SelectivePurification of Glycoproteins and Glycoforms Displaying Terminalβ1,4-Linked Galactose. (A) SDS-PAGE analysis of fractions obtained fromlectin pull down assays performed using rPA-ILNmE6 Sepharose. Lane (1) 4μg asialotransferrin; (2) 4 μg carbonic anhydrase; (3) 4 μg glucoseoxidase; (4) 4 μg cytochrome C; (5) molecular weight ladder (NEB widerange protein ladder); (6) 20 μL of a protein mix containing 200 μg mL⁻¹of each of the proteins shown in lanes 1 to 4; (7) 20 μL of unboundprotein fraction and (8) 20 μL bound galactose eluted protein fraction.It can be clearly seen that the asialotransferrin was successfullyisolated from the protein mixture by the rPA-ILNmE6 Sepharose resin. (B)Separation of glycoprotein glycoforms: a 2 mL sample comprised of amixture of 1 mg of transferrin and 1 mg of asialotransferrin was appliedto a 1 mL rPA-ILNmE6 Sepharose FPLC column. The sample was effectivelyseparated into two fractions: one fraction comprised of unbound protein(U) and one comprised of bound protein (Bd) which was selectively elutedby inclusion of 0.5 M galactose in the mobile phase. (C) ELLA analysisof FPLC fractionated transferrin glycoforms. Only the bound fractionelicited strong responses from the galactophilic lectins confirmingeffective separation and isolation of the asialotransferrin glycoforms.

FIG. 8: Expression Vectors Constructed for Expression of rPA-ILProteins: Panels (A) and (B) show maps of the constructed pQE30PA-IL andpQE60PA-IL vectors respectively. Panels (C) and (D) show the codingregions of the pQE30PA-IL and pQE60PA-IL expression vectors. Importantsequence elements highlighted: the ribosome binding site (RBS), startand stop codons (bold underlined); restriction sites (bold shaded);residues subjected to random mutagenesis (bold italics underlined).Sequences primed for mutagenesis are indicated by arrows; PA-ILmutF(dashed arrow) and PA-ILmutR (solid arrow). Panel (E) The amino acidsequences of rPA-ILN and rPA-ILC. The pQE30PA-IL vector expresses amature rPA-ILN protein of 133 amino acids (excluding the initiatormethionine) with an estimated molecular weight of 14.16 kDa, pl of 6.45and extinction coefficient of 1.974. The pQE60PA-IL vector expresses amature rPA-ILC protein of 129 amino acids (excluding the initiatormethionine) with an estimated molecular weight of 13.83 kDa, a pl of6.45 and extinction coefficient of 2.02. Residues targeted formutagenesis are highlighted in bold underlined.

FIG. 9: SDS-PAGE Analysis of PA-IL Proteins: Panels (A) and (B) showSDS-PAGE analysis of samples from the expression and purification ofrPA-ILC and rPA-ILN respectively. In each panel: Lane 1—molecular weightladder (NEB Wide Range Protein Ladder—sizes are in kDa); Lane 2—solublecell lysate (CL); Lane 3—final sample of flow through cell lysate (FT);Lane 4—final sample of 80 mM imidazole wash; Lane 5—eluted rPA-ILprotein. The rPA-IL proteins (indicated by arrows) ran around 14 kDa asexpected. High level expression of both of the rPA-IL proteins in thesoluble cell lysate fraction can be clearly seen. Both proteins wereeffectively captured by IMAC resin resulting in no rPA-IL bands beingvisible in FT fractions or in high stringency 80 mM imidazole washfractions. Both of the purified rPA-IL proteins exhibited a very highlevel of purity. (C) Comparison of purified rPA-IL proteins withcommercial untagged PA-IL (PA-ILU). Lane 1—SDS-PAGE protein standards;Lane 2—PA-ILU; Lane 3—rPA-ILN; Lane 4—rPA-ILC. All proteins ran at theirexpected molecular weights with rPA-ILN and rPA-ILC running slightlylarger (14.1 kDa and 13.8 kDa respectively) than PA-ILU (12.7 kDa).

FIG. 10: GPC Analysis of PA-ILU, rPA-ILC and rPA-ILN: GPC was conductedusing a Superdex 75 10/300 GL column (GE Healthcare) which had a totalvolume (V_(t)) of 19 mL and a void volume (V_(o)) of 7.73 mL.Experiments were performed using an AKTA purifier and all samples wererun in PBS and therefore under physiological conditions. Elution volumes(V_(e)) were experimentally determined for a series of molecular weightstandards. This enabled the calculation ofK_(ay)[(V_(t)−V_(e))/(V_(t)-V₀)]values for each of the protein standardsand the subsequent preparation of a plot of molecular weight (MW) versusK_(av) which had an R² value of 0.997. This plot was used to calculatean estimated molecular weight (MW_(e)) for commercially obtaineduntagged PA-IL protein (PA-ILU) which is known to be a tetramer atphysiological pH with a molecular weight of 51 kDa (23). The GPCestimated molecular weight of 41.5 kDa was significantly less than theexpected 51 kDa. The discrepancy between the actual and theexperimentally determined molecular weights for the PA-ILU protein islikely to be due to conformational differences between the PA-ILUprotein and the globular proteins used as standards to generate theK_(av) plot. The rPA-ILC protein had an estimated molecular weight of40.3 kDa, which was similar to that of the PA-ILU protein, and it wastherefore determined to be tetrameric. The rPA-ILN protein was estimatedto have a molecular weight of 19.6 kDa, approximately half that of therPA-ILC protein, and it was therefore proposed to be a dimer.

FIG. 11: Functional Analysis of the rPA-IL Proteins Using theHemagglutination Assay. (A) Hemagglutination Assays: In each panel, well1 is a negative control to which no lectin was added. For the PA-ILU andPA-ILC proteins, well 2 had a final lectin concentration of 25 μg mL⁻¹while, for the rPA-ILN protein, the concentration was 50 μg mL⁻¹.Subsequent wells had serial 1:2 dilutions of the lectins. Both thePA-ILU and rPA-ILC showed similar hemagglutination profiles displayingfull agglutination up to well 9 (195 ng mL⁻¹). The rPA-ILN showed asignificantly decreased capacity to agglutinate RBC's with completeagglutination visible up to well 5 (6.25 μg mL⁻¹). For the PA-ILU andrPA-ILC proteins 1 HU was therefore calculated to be 195 ng mL⁻¹ while,for rPA-ILN, it is was calculated to be 6.25 μg mL⁻¹. (B)Hemagglutination Inhibition assays: Inhibitions were carried out withthree carbohydrates; raffinose (Raf: Gal-α1,6-Glc-β1,2-Fru), melibiose(Mel: Gal-α1,6-Glc) and galactose (Gal) known to be bound by the PA-ILprotein (28). In each panel: Lane 1—negative control with no lectinadded; Lane 2—positive control containing 2HU of each lectin and noinhibiting carbohydrate. For raffinose and melibiose inhibitions, well 3had a final carbohydrate concentration of 2.5 mM while, for galactose,it was 6.25 mM. Subsequent wells contained a serial 4:5 dilution seriesof the carbohydrate. Both the PA-ILU and rPA-ILC proteins showed similarinhibition profiles for all carbohydrates. For both raffinose andmelibiose inhibitions, complete inhibition was scored to well 8 (0.82mM). Galactose was less effective, with full inhibition being scored towell 7 (2.56 mM). The rPA-ILN protein was significantly more sensitiveto inhibition with all carbohydrates tested. Melibiose and raffinosewere found to completely inhibit in wells 16 (0.18 mM) while galactosewas found to fully inhibit in well 19 (0.14 mM).

FIG. 12: Functional Analysis of rPA-IL Proteins by ELLA. (A) TherPA-ILC, rPA-ILN and GSL-I lectins were tested for their ability to binda BSA-αGal glycoconjugate by ELLA. The glycoconjugate was immobilized ata concentration of 5 μg mL⁻¹ and each of the lectins was evaluated overa range of concentrations (5 μg mL⁻¹ to 50 ng mL⁻¹). Biotinylated GSL-Iwas detected using an anti-biotin antibody, while bound rPA-IL proteinswere detected using an anti-HIS antibody. Of the two rPA-IL proteins,only binding of the rPA-ILN protein could be detected. As rPA-ILC hadbeen demonstrated to be functional via other methods, the failure ofthis protein to generate signals in ELLA's was due to stericunavailability of the 6HIS tag to binding by the anti-HIS antibody.

FIG. 13: Qualitative ELLA analysis of rPA-ILNm proteins carrying His50substitutions. The rPA-ILNm mutants are labelled with a single lettercode indicative of the His50 substitution they carry. The rPA-ILNprotein (WT) and certain of the rPA-ILNm proteins from Example A wereincluded for comparative purposes: rPA-ILNmE6 (E6), rPA-ILNmC5 (C5),rPA-ILNmF3 (F3), rPA-ILNmB4 (B4). A number of plant lectins were alsoincluded as controls; ECL (β1,4-linked galactose), GSLI (α-linkedgalactose), RCA (galactophilic and tolerates capping α2,6 sialic acid),SNA (α2,6 sialic acid) and MALII (α2,3 sialic acid). These confirm theabsence of capping sialic acid on asialotransferrin. Results clearlyidentify a number of His50 substitutions that alter the carbohydratebinding specificity and affinity compared to the rPA-ILN protein.Proteins displaying significant binding to the BSA-LacNAc and BSA-αGalglycoconjugates are indicated by black and white arrows respectively.

FIG. 14: Lectin dose response curves for selected His50 substitutedrPA-ILNm proteins. (A) Response curves for selected lectins testedagainst BSA-LacNAc. Results clearly show the rPA-ILNmE6 (E6) exhibitsthe highest affinity but this is only slightly greater than thatdisplayed by H50N (N) and H50E (E). H50V (V), H50Q (Q) and H50T (T)proteins displayed significantly and progressively weaker bindingaffinities to this glycoconjugate. (B) Response curves for selectedlectins tested against BSA-αGal. Results clearly show that the H50Qprotein exhibited the highest affinity for this glycoconjugate. Althoughthis was greater than its affinity for the BSA-LacNAc glycoconjugate, itwas still significantly weaker than that of the parental rPA-ILN protein(WT). The H50K (K) protein displayed a slightly lower affinity than thatof the H50Q. The H50N protein also bound to the BSA-αGal glycoconjugateand, while lower than that of H50Q and H50K, it was significantlygreater than that displayed by the rPA-ILNmE6 protein.

FIG. 15: Qualitative ELLA analysis of H50N proteins with additionalGln53 substitutions. The rPA-ILNm proteins tested are represented by atwo letter code indicating the amino acid substitutions they carry e.g.NG has H50N and Q53G substitutions. The rPA-ILN protein (WT), rPA-ILNme6(E6) and H50N (N) were included for comparative analysis. Results showthat substitution of the Gln53 residue with a range of different aminoacids only resulted in subtle differences in binding activities towardsthe two BSA glycoconjugates and the asialotransferrin (AsT). None of thenew H50N:Q53 double mutants bound to the BSA-LacNAc or asialotransferrinbetter than the H50N. Interestingly, the NG protein generated weakersignals on asialotransferrin than the H50N and significantly weaker thanthose generated by the rPA-ILNmE6 protein. The H50N:Q53E proteinexhibited strong signals on the BSA-αGal glycoconjugate which werecomparable to, if not slightly stronger than, those of the parental H50Nprotein but significantly lower than those obtained for the rPA-ILNprotein. However unlike the H50N protein it exhibited negligible bindingtowards asialotransferrin.

FIG. 16: The role of a Q53R substitution in promoting binding toα-linked galactose: (A) Qualitative ELLA screen of selected rPA-ILNmproteins carrying Q53R, Q53K or Q53E substitutions. The rPA-ILN protein(WT), rPA-ILNmE6 (E6) and rPA-ILNmF3 proteins were included forcomparative analysis. The rPA-ILNm proteins tested are represented by atwo letter code indicating the amino acid substitutions they carry inplace of His50 and Gln53 respectively. Results clearly show thatintroduction of a Q53R substitution into H50V and H50Q (generating VR,and QR respectively) resulted in a slight increase in signals againstBSA-αGal proteins. The H50V:Q53R protein also displayed increasedbinding to terminal β1,4 linked galactose compared to the H50V proteinwhich was in contrast to the H50Q:Q53R protein which exhibited asignificant reduction in binding to β1,4 linked galactose compared tothe H50Q protein. When a single Q53R substitution was introduced intothe parental rPA-ILN protein it again resulted in enhanced binding toBSA-αGal and this was also observed for a conservative Q53E substitution(HR and HE proteins respectively). (B) Lectin dose response curves forselected rPA-ILN proteins with Q53R or Q53E substitutions. Also includedare the rPA-ILN (WT), rPA-ILNmF3 (F3) and H50Q proteins for comparativepurposes. It can be seen that rPA-ILNm proteins carrying either a singleQ53E (HE) or Q53R (HR) substitution exhibited higher relative bindingaffinities for the BSA-αGal glycoconjugate than the parental wild typerPA-ILN protein. The H50Q:Q53R (QR) protein also exhibited a higheraffinity than its parental H50Q (Q) protein. While a parental H50Vprotein was not observed to bind significantly to the BSA-αGalglycoconjugate, it can be seen that the H50V:Q53R protein exhibited anaffinity for the glycoconjugate comparable to that of the rPA-ILNmF3protein.

FIG. 17: Structural models depicting the probable steric role played bythe amino acid at position 50 in determining the linkage specificity ofPA-IL proteins. (A) PA-IL in complex with iGb3. (B) PA-IL with lactosemodelled into the binding pocket such that the terminal galactose moietyis bound in the configuration observed in crystal structures obtained todate with either bound D-galactose (29) or iGb3 (23). This illustratesthat binding of the terminal galactose of a lactose molecule in thisconfiguration would result in the glucose moiety sterically clashingwith the His50 residue. (C, D & E). Models showing the possible effectsof H50N, H50V and H50Q mutations, respectively, on the conformation ofthe PA-IL binding site. ELLA analysis demonstrated that replacing His50with these amino acid residues promoted binding of glycans with terminalβ1,4-linked galactose. Models suggest this is partly the result of amore open conformation in the binding pocket thereby allowing glycanswith β-linked galactose to enter.

FIG. 18: Structural models of rPA-ILNm lectins with defined His50substitutions and bound lactose (Gal-β1,4-Glc). (A) H50N: Hydrogen bondsbetween the Asn50 side chain and both sugar residues may occur. The sidechain of Tyr36 may also form a hydrogen bond, with the galactose moietyto stabilize binding to p-linked galactose in each of the HSO mutants.(B) H50V: Va150 cannot form hydrogen bonds with the sugar, although itmay form some stabilizing hydrophobic contacts with the glucose moiety.(C) H50Q: It is difficult to predict if a glutamine residue will makecontacts with a bound sugar as the side chain may adopt severalorientations. However, ELLA results show that this protein is capable ofbinding strongly to BSA-LacNAc, and the side chain orientation depictedshows that it may enable hydrogen bonding with the substrate. (D) Randommutant rPA-ILNmE6. The Q53G substitution would potentially result in theloss of the hydrogen bond between Gln53 and the terminal galactose sugarmoiety.

FIG. 19: Structural models of rPA-ILNm mutants with iGb3(Gal-α1,3-Gal-β1,4-Glc). (A) H50N, (B) H50V, (C) H50Q and (D)rPA-ILNmE6. The H50Q substitution showed the highest affinity of all ofthe His50 mutants for BSA-αGal in ELLA's. This is potentially due to theformation of an additional hydrogen bond with the second galactose inthe oligosaccharide chain. An Asn50 side chain, also found inrPA-ILNmE6, can possibly interact with the terminal galactose, whileVal50, which shows the lowest binding to BSA-αGal, does not makepositive contacts with the sugar.

FIG. 20: Amino Acid Sequences of the rPA-ILN Protein and Derived Mutants(rPA-ILNm). Residues incorporated at the N-terminus (the 6HIS affinitypurification tag and additional amino acid residues) are boxed. Thenatural initiator methionine of the PA-IL protein is indicated in blackbold. Residues randomly substituted in mutants are bold and underlined.Please note:

-   -   a. These correspond to residues His62, Aps64 and Gln65 in the        rPA-ILN protein excluding its initiator methionine; or    -   b. These correspond to residues His50, Asp52 and Gln53 in the        wild type PA-IL protein excluding its initiator methionine.

The abbreviations used are: PA-IL, Pseudomonas aeruginosa lectin 1;rPA-IL, recombinant PA-IL; ELLA, enzyme linked lectin assay; iGb3,isoglobotriaosylceramide (Gal-α1,3-Gal-β1,4-Glc); PBS, PhosphateBuffered Saline; TBS, Tris Buffered Saline; TBST, Tris Buffered Salinewith Tween 20; IPTG, Isopropyl-β-D-thiogalactopyranoside.

The glycoconjugate used as a representative of glycoproteins displayingglycans with terminal β-linked galactose was BSA-LacNAc(Gal-β1,4-GlcNAc-BSA). Lectins representative of those showing bindingto a terminal β-linked galactose include ECL (Erythrina cristagalliLectin) and RCA (Ricinus communis Agglutinin).

The glycoconjugate used as a representative of glycoproteins displayingglycans with terminal α-linked galactose was BSA-αGal(Gal-α1,3-Gal-BSA). The lectin used as a representative of those showingbinding to a terminal α-linked galactose was GSLI (Griffoniasimplicifolia isolectin B4).

EXAMPLE A Experimental Procedures

Plasmid Construction—pQE30PA-IL & pQE60PA-IL—All strains and plasmidsused or constructed as part of this study are listed and described inTable A1 set out below. The lecA gene encoding the PA-IL protein wasamplified from Pseudomonas aeruginosa PAO1 (This strain can be obtainedfrom a wide variety of sources including many cell culture banks)genomic DNA by PCR to facilitate cloning into the pQE series of E. coliexpression vectors from Qiagen. PCR reactions were carried out usinghigh fidelity Phusion Taq and PCR conditions recommended by themanufacturer (New England BioLabs). The lecA gene was amplified usingthe PA-IL-F1 and PA-IL-R1 primers (Table A2 below) to generate a productthat could be cloned as a BamHI-HindIII fragment into the pQE30expression vector. The resulting plasmid, pQE30PA-IL (FIG. 8A),expressed an rPA-IL protein with an amino (N-) terminal 6HIS tag(rPA-ILN). The lecA gene was also amplified using the PA-IL-F2 andPA-IL-R2 primers to enable cloning as an Ncol-BgIII fragment into thepQE60 vector (Qiagen). The resulting plasmid, pQE60PA-IL (FIG. 8B),expressed an rPA-IL protein with a carboxy (C-) terminal 6HIS tag(rPA-ILC).

TABLE A1 Strains and Plasmids used in Example A. Strains GenotypeDescription Source Escherichia coli JM109 F′traD36, proAB+ lacI^(q),ΔlacZ M15, endA1, All purpose cloning Promega recA1, hsdR17(r_(k)−,m_(k)+), mcrA, supE44, λ- strain, produces stable gyrA96, relA1Δ(lacproAB), thi-1. plasmid DNA. KRX [F′, traD36, ΔompP, proA+B+,lacI^(q), Protease deficient Promega (30) Δ(lacZ)M15] ΔompT, endA1,recA1, gyrA96 protein expression (Nalr), thi-1, hsdR17 (r_(k)−, m_(k)+),relA1, host. supE44, Δ(lac-proAB), Δ(rhaBAD)::T7 RNA polymerase.Pseudomonas aeruginosa PAO1 Wild Type Dr. Keith Poole Plasmids FeaturesDescription Source pQE30 T5 promoter/lac operator element, rrnBExpresses proteins with Qiagen T1 transcriptional termination region, anN-terminal RGS- ColE1 origin, β-lactamase gene. 6HIS affinity tag. pQE60T5 promoter/lac operator element, rrnB Expresses proteins with Qiagen T1transcriptional termination region, a C-terminal 6HIS ColE1 origin,β-lactamase gene. affinity tag. pQE30PA-IL pQE30 with cloned P.aeruginosa lecA Expresses rPA-ILN: Example A Wild type PA-IL with anN-terminal RGS- 6HIS affinity tag. PQE60PA-IL pQE60 with cloned P.aeruginosa lecA Expresses rPA-ILC: Example A Wild type PA-IL with aC-terminal 6HIS affinity tag. Mutagenized Amino Acid Protein PlasmidsSubstitutions Expressed Source pPC30PA-IL-A8 H50L D52H Q53R rPA-ILNmA8Example A pPC30PA-IL-B4 H50T D52N Q53R rPA-ILNmB4 Example ApPC30PA-IL-B10 H50V D52C Q53E rPA-ILNmB10 Example A pPC30PA-IL-C5 H50ND52T Q53S rPA-ILNmC5 Example A pPC30PA-IL-E6 H50N D52N Q53G rPA-ILNmE6Example A pPC30PA-IL-E12 H50G D52C Q53R rPA-ILNmE12 Example ApPC30PA-IL-F3 H50V D52C Q53R rPA-ILNmF3 Example A pPC30PA-IL-F6 H50PD52R Q53L rPA-ILNmF6 Example A pPC30PA-IL-G3 H50V D52N Q53N rPA-ILNmG3Example A

TABLE A2 Primer Sequences used in Example APrimers Used for Amplification and Cloning of the lecA Gene into pOE Expression VectorsForward Primers PA-IL-F1 AaaaGGATCC atggcttggaaaggtgagg - SEQ ID NO. 42PA-IL-F2 aaaaCC ATG Gcttggaaaggtgaggttctgg - SEQ ID NO. 43Reverse Primer PA-IL-R1 aaaaAAGCTTtcacgactgatcctttccaatatt - SEQ ID NO. 44 PA-IL-R2aaaaAGATCTcgactgatcctttccaatattgacac - SEQ ID NO. 45Primers Used for Site Specific Mutagenesis of the lecA Gene.Forward Primer PA-ILmutFcgttttgtggtgcgctggtcatgaagattggc - SEQ ID NO. 46Reverse Primer Used for Random Mutagenesis of H50, D52 and Q53 PA-ILmutR

Protein Expression and Purification—For protein expression plasmids weretransformed into the protease deficient E. coli strain KRX (30).Expression clones were cultured at 30° C. in Terrific Broth (TB) brothand protein expression induced by addition of IPTG to a finalconcentration of 50 μM. Cells were harvested by centrifugation and cellpellets resuspended in lysis buffer [10 mM NaH₂PO₄, 300 mM NaCl, 40 mMimidazole, pH 8.0). Cell disruption was achieved by high pressure usinga Constant Systems™ cell disrupter and cell debris was removed bycentrifugation. Clarified cell lysates were applied to 10 mL IMAC™columns (IMAC Hypercel from Pal) and a high stringency wash buffer with100 mM imidazole was used to remove non-specifically bound contaminatingproteins. The desired 6HIS tagged proteins were ultimately eluted using250 mM imidazole and eluted proteins were aliquoted and stored at −80°C. in the elution buffer. Typical yields were around 200 mg per 250 mLstarting culture. Purified proteins were analysed by SDS-PAGE to assesspurity (FIG. 9) and routinely buffer exchanged and concentrated usingVivaspinTM centrifugal membrane devices (Sartorius-Stedim), with amolecular weight cut off of 10 kDa, according to the manufacturer'sguidelines.

Gel Permeation Chromatography (GPC)—The estimated molecular weights ofthe rPA-IL proteins were determined by GPC, which was performed on aSuperdex™ 75 10/300 GL column (GE Healthcare) using an AKTA Purifier 100FPLC system. The molecular weight of commercially obtained untaggedPA-IL (Sigma Aldrich) was also experimentally determined and used forcomparison with 6HIS tagged rPA-IL proteins to enable determination oftheir quaternary structure (FIG. 10).

Hemagglutination Assays—The hemagglutination assay is widely used tostudy lectin activity and is dependent on the multi-valency typicallydisplayed by lectins. The assay was essentially performed according tothe method described by Garber et al (31). The assay was performed usingPapain treated Rat red blood cells (RBC's), obtained from theBioresource unit at DCU such that the final concentration of cells inreaction wells was 3.5% w/v. Lectins to be tested were prepared in TBS(20 mM Tris, 150 mM NaCl, 1 mM CaCl₂, 1 mM MnCl₂, 1 mM MgCl₂, pH 7.6).Hemagglutination was observed, after 1 hour incubation at 25° C., as athin film of cells coating the bottom of wells in U-bottomed 96 wellplates compared to a concentrated spot of sedimented cells observed innegative controls to which no lectin was added (FIG. 11A). Onehemagglutination unit (HU) was defined as the minimum quantity of lectinrequired to fully agglutinate the RBC solution. Sugar inhibition assayswere performed such that the final lectin concentration in reactionwells was equivalent to 2 HU (FIG. 11B).

General Enzyme Linked Lectin Assay (ELLA) Method—The Gal-α1,3-Gal-BSA(BSA-αGal) and Gal-β1,4-GlcNAc-BSA (BSA-LacNAc) glycoconjugates usedwere from Dextra Laboratories. Biotinylated plant lectins GSLI(Griffonia simplicifolia isolectin B4), ECL (Erythrina cristagalliLectin), RCA (Ricinus communis Agglutinin), SNA (Sambucus nigraAgglutinin), MALII (Maackia amurensis Lectin) and LCA (Lens culinarisAgglutinin) were from Vector Laboratories. The glycoproteins fetuin,asialofetuin and invertase were from Sigma Aldrich whileasialotransferrin, agalactotransferrin and agalactofetuin were generatedby treatment using glycosidases, neuraminidase (Clostridium perfingens)and β1,4-galactosidase (Bacteroides fragilis), in accordance withmanufacturer's guidelines (New England Biolabs). ELLA's were essentiallyperformed according to the method described by Thompson et al (2011)(33). More specifically, glycoproteins were prepared in PBS andtypically immobilized at a concentration of 5 μg mL⁻¹. For qualitativeELLA's, lectins were assayed at a concentration of 10 μg mL⁻¹ in TBST(20 mM Tris, 150 mM NaCl, 0.05% Tween-20, 1 mM CaCl₂, 1 mM MnCl₂, 1 mMMgCl₂, pH 7.6). For lectin dose response experiments, each lectin wasevaluated at a range of concentrations prepared by serial 1:2 dilutionof an initial lectin solution of 10 μg mL⁻¹ to a final concentration of156 ng mL⁻¹. Binding of 6HIS tagged rPA-IL proteins was detected after 1hour incubation at 25° C. using a HRP conjugated anti-HIS antibodydiluted 1:10,000 in TBST (Sigma Aldrich). Biotinylated plant lectinswere detected using a HRP conjugated anti-biotin antibody diluted1:10,000 in TBST (Sigma Aldrich).

Lectin Affinity Constant Determination by ELLA—Affinity constants weredetermined according to the method described by Kirkeby et al 2002 (27).ELLA's were performed using a constant concentration of 2 μg mL⁻¹ forthe rPA-ILNm proteins and 4 μg mL⁻¹ of ECL to ensure all lectins wereevaluated at equimolar concentrations. Each lectin was evaluated againstBSA-LacNAc glycoconjugate immobilized at a range of concentrations from10 μg mL⁻¹ to 19.5 ng mL⁻¹ (prepared by serial 1:2 dilution of a 10 μgmL⁻¹ stock). The resulting glycoconjugate dose response curve obtainedenabled the calculation of B_(max) and the affinity constant K_(D) foreach lectin against BSA-LacNAc. B_(max) is defined as being the maximumplateau value of absorption and represents the maximum number of lectinbinding sites expressed in the units of the Y-axis (AU). K_(D) isdefined as being the glycoconjugate concentration required to fill halfof the available lectin binding sites at equilibrium. K_(D) is thereforethe glycoconjugate concentration that generates a signal equivalent tohalf B_(max) and the unit for K_(D) is nanograms of glycoconjugate.These values are specific for the defined experimental conditions used.

Site Directed Random Mutagenesis of the rPA-ILN Protein—PCR based sitedirected mutagenesis of the lecA gene was achieved through whole vectoramplification in which the pPC30PA-IL vector was used as the parentaltemplate DNA. Whole vector amplification was achieved using the primersPA-ILmutF and PA-ILmutR (Table A2 above). These primers were 5′phosphorylated and designed to anneal within the lecA sequence withtheir 5′ ends exactly next to each other. The reverse primers weredesigned to overlap the region to be mutagenized enabling theintroduction of mutations through manipulation of reverse primersequences (FIG. 8C). Successful PCR reactions were purified andsubjected to digestion with the restriction enzyme DpnI to selectivelydigest the parental template vector DNA. Digestions were ultimately runon agarose gels and PCR products corresponding to the expected size oflinear vector were gel extracted. The final purified PCR products, withblunt phosphorylated ends, were re-circularised by simple self ligationand the ligated DNA transformed into E. coli strain KRX. Transformantswere picked into sterile deep well (2 mL) 96 well plates to generatemaster arrays of clones capable of expressing mutated rPA-ILN proteins(rPA-ILNm). Master plates were used to inoculate fresh 96 well platescontaining 600 μL of LB media supplemented with ampicillin (100 μg mL⁻¹)and IPTG (50 μM) to induce expression of rPA-ILNm proteins. Afterovernight incubation at 30° C. cells were harvested by centrifugationand subsequently disrupted chemically by resuspending cell pellets in 1×Cell Lytic B solution (Sigma Aldrich) by repeated gentle pipetting.Plates were then incubated at room temperature for 1 hour or overnightat 4° C. to allow disruption of cells. Cell debris was removed bycentrifugation and cell lysates were diluted 10 fold using TBST prior tobeing used in ELLA screens.

Fabrication of Lectin Affinity Resins—lectin affinity resins wereprepared by immobilization onto cyanogen bromide (CNBr) activatedSepharose 4B prepared according to the manufactures guidelines (GEHealthcare). Lectin to be immobilized was buffer exchanged into couplingbuffer (20 mM NaH₂PO₄, 500 mM NaCl, pH 8.5) and the lectin solutionmixed with the CNBr Sepharose at a concentration of approximately 30 mgmL of resin. This mixture was then left mixing by inversion overnight at4° C. Unbound protein was then decanted and un-reacted CNBr groups onthe resin were capped with 1 M ethanolamine in coupling buffer and pH8.5. This was left mixing by inversion at room temperature for 4 hours.Finally non-specifically bound protein was removed by 4 successivewashes with coupling buffer and actetate buffer (100 mM NaOAc, 500 mMNaCl, pH 4.0). Resins were ultimately washed with TBS and for long termstorage sodium azide was added to a final concentration of 2 mM.

Evaluation of Lectin Affinity Resins—lectin affinity resins wereevaluated by performing small scale pull down assays in 1.5 mL eppendorftubes or by packing into 1 mL FPLC cartridges (FIiQ Column Housings,Generon) to enable easy connection to FPLC systems. For pull downassays, 50 μL of lectin affinity resin was mixed with 100 μL of a testprotein mixture and mixed by inversion for 1 hour. Unbound protein wasthen removed using a pipette and the resin washed with three 1 mLaliquots of TBST. Bound protein was eluted by addition of 100 μL of TBSTwith 0.5 M galactose followed by incubation for 1 hour. Lectin affinityFPLC columns were connected to an AKTA Purifier 100 FPLC system. Columnswere equilibrated using TBS and typically run at a flow rate of 0.5 mLper minute. Samples were prepared in TBS and 2 mL sample volumes wereinjected onto the 1 mL lectin affinity columns. Bound glycoproteins wereeluted using 0.5M galactose prepared in TBS.

RESULTS

Production of Affinity Tagged Recombinant PA-IL (rPA-IL)—Commerciallyavailable untagged PA-IL (PA-ILU) is typically purified by exploitingits natural affinity for Sepharose 4B (23,34) but alteration of theprotein's carbohydrate binding specificity could prevent purification inthis manner. One of the first steps required for this study was,therefore, the incorporation of an affinity tag that would enable simpleand rapid purification of recombinant PA-IL (rPA-IL) proteinsindependently of glycan binding specificity. The lecA gene, encoding thewild type PA-IL protein, was therefore cloned into two E. coli plasmidexpression vectors to enable expression of rPA-IL with either anN-terminal (rPA-ILN) or C-terminal (rPA-ILC) polyhistidine (6HIS) tagand thereby enabling purification by IMAC. Both proteins were expressedto relatively high levels in the soluble cytoplasmic fraction of E. coliKRX, from which they were subsequently purified by IMAC. Assessment ofthe proteins by SDS-PAGE verified that both exhibited very high levelsof purity (FIG. 9) and yields were typically around 800 mg L⁻¹ ofculture.

Structural and Functional Assessment of Poly-Histidine Tagged rPA-ILProteins—Incorporation of a poly-histidine tag into any protein can havean impact on the structure, activity and other physiochemical properties(solubility, stability) of the protein. We therefore assessed the impactof the incorporated 6HIS tags on the quaternary structure andfunctionality of the rPA-IL proteins. Gel permeation chromatography(GPC) was used to experimentally determine the molecular weight ofcommercially obtained untagged PA-IL (PA-ILU), which is known to be atetramer of four identical subunits under physiological conditions(FIG. 1) (23,29). This was used as a reference for comparison with therPA-IL proteins to determine their quaternary structure. The rPA-ILCprotein was determined to be a tetramer, like the PA-ILU protein, butthe rPA-ILN protein was determined to exist as a dimer, indicating thatthe incorporation of the 6HIS affinity tag at the N-terminus of theprotein had disrupted the quaternary structure of the protein to someextent (FIG. 10). The functionality of the rPA-IL proteins was assessedand compared to that of the PA-ILU protein using the hemagglutinationassay. The rPA-ILC protein was found to have comparable activity toPA-ILU in hemagglutination assays with both proteins fully agglutinatingPapain treated rat red blood cells (RBC's) at a lectin concentration of195 ng mL⁻¹ (FIG. 11A). Both proteins also showed comparable inhibitionprofiles for all sugars assessed in sugar inhibition assays (FIG. 11B).The rPA-ILN protein was significantly less effective at agglutinatingRBC's, requiring a concentration of 6.25 μg mL⁻¹ to produce fullagglutination. This was also more readily inhibited by all of the sugarsevaluated in sugar inhibition assays. Hemagglutination assays confirmedthat both of the rPA-IL proteins were functional and that differences intheir comparative performance reflected the differences in theirrespective quaternary structures where the rPA-ILN protein only has halfthe valency of the rPA-ILC protein. The ability of the rPA-ILN proteinto agglutinate RBC's also provided evidence that the quaternarystructure of the rPA-ILN protein was likely to be that of a dimer withan A-D subunit configuration (FIG. 1) because a dimer with an A-Bsubunit configuration would be unlikely to be capable of cross linkingcell surfaces.

Selection of a Target rPA-IL Molecule for Mutagenesis Studies—The stericaccessibility of the 6HIS tags within the quaternary structures of therPA-IL proteins was assessed by ELLA. Both of the rPA-IL proteins weretested for their ability to bind an immobilized BSA-αGal glycoconjugatewith subsequent detection of the bound lectins using an anti-HISantibody (33). Biotinylated GSL-I, a plant lectin with a bindingspecificity for terminal α-galactose (27,35), was included in the assaysas a positive control. Binding of both rPA-ILN and GSLI could bedetected in ELLA's with high sensitivity but binding of the rPA-ILCcould not be detected using an anti-HIS antibody, even at relativelyhigh lectin concentrations of 10 μg mL⁻¹ (not shown in FIG. 12A). As thefunctionality of both of the rPA-IL proteins had already been confirmed,this was therefore due to steric unavailability of the 6HIS tags withinthe rPA-ILC tetramer to binding by the anti-HIS antibody. This wasfurther confirmed by direct immobilization of the rPA-ILC protein inELISA plates and probing with anti-HIS antibody which failed to generatesignals (data not shown). While incorporation of a 6HIS tag at theN-terminus of the rPA-IL was shown to disrupt the natural tetramericstructure of the PA-IL protein, the resulting dimeric molecules weredemonstrated to be functional and could be readily detected, with highsensitivity, in ELLA's using an anti-HIS antibody. The pQE30PA-IL vectorwas therefore selected as the target DNA molecule for mutagenesisbecause the production of mutagenized rPA-IL proteins with N-terminallypositioned 6HIS tags (rPA-ILNm) would not only facilitate their simplepurification but also enable analysis of their carbohydrate bindingactivities through the use of ELLA assays without the need for any priorin vitro labelling steps. As all of the mutagenized proteins wouldultimately be compared to the parental rPA-ILN protein, and wouldtherefore have equivalent quaternary structures, comparative analysiscould be performed by ELLA to identify proteins with alteredcarbohydrate binding properties.

Random Mutagenesis of the rPA-ILN Protein—Residues from three separateparts of the PA-IL monomer are involved in coordinating calcium andbinding of the iGb3 trisaccharide Gal-α1,3-Gal-β1,3-Glc (23) (FIG. 1).Amino acid residues involved in the coordination of the essentialcalcium ion were not selected for mutagenesis since alteration of theseresidues would likely result in loss of calcium coordination andconsequently result in loss of carbohydrate binding. As residues His50and Gln53 are only involved in making contacts with the carbohydrate,these residues were selected as target amino acid residues formutagenesis. The intervening Pro51 and Asp52 residues were not thoughtto be directly involved in carbohydrate binding but modification ofthese residues could alter the spatial arrangement of the His50 andGln53 residues relative to each other in the binding site andconsequently impact carbohydrate binding. Proline residues introducekinks and rigidity into polypeptide strands. Alteration of this residuemight therefore be expected to result in dramatic structural changes inthe carbohydrate binding site to negatively impact carbohydrate bindingand so this residue was not selected for mutagenesis. However,substitution of the Asp52 residue might have more subtle affects and sothis residue was selected for inclusion in the mutagenesis study.

The pQE30PA-IL plasmid was mutagenized using a PCR based method thatresulted in the introduction of random simultaneous substitutions atpositions corresponding to residues His50, Asp52 and Gln53 in the wildtype native PA-IL protein. An array of mutant clones, containing 154individual mutants, was prepared and cell lysates from these clones wereanalysed by ELLA. A number of different glycoproteins were used asimmobilized targets in ELLA screens to identify clones expressingmutated rPA-ILN proteins (rPA-ILNm) exhibiting altered bindingspecificities compared to the parental rPA-ILN protein. Glycoproteinstested included fetuin (3 N-linked and 3 O-linked glycan structureshighly sialylated with terminal α2,3 and α2,6-Neu5Ac) (36), asialofetuin(terminal LacNAc and terminal β1,3-Gal) and invertase (high mannosestructures) (37). PA-IL is known not to bind strongly to theseglycoprotein targets (28) and so any rPA-ILNm proteins identified asgenerating altered responses to these targets were considered to havealtered carbohydrate binding properties. Of the 154 clones screened,none of the rPA-ILNm proteins displayed binding to invertase orsignificant binding to fetuin but a number of rPA-ILNm proteins wereobserved to exhibit altered binding to asialofetuin (data not shown).Nine of the rPA-ILNm proteins that exhibited the highest signals againstthese glycoproteins in the ELLA screen were selected and recovered fromthe array for further analysis. The selected rPA-ILNm proteins werenamed according to the well from which they were recovered in theoriginal mutant array (i.e. rPA-ILNmE6 was recovered from row E andcolumn 6).

The initial ELLA mutant library screen had been performed using solublefractions of cell lysates and therefore differences observed in ELLAresponses may have been impacted by differences in the expression levelsof specific rPA-ILNm proteins or differences in the overallconcentrations of cell lysates. To validate the results of the initialELLA screen, the selected rPA-ILNm proteins were first purified by IMACand then re-evaluated in qualitative ELLA's against an expanded set ofglycoproteins and specifically generated glycoprotein glycoforms (FIG.2). Transferrin is a commercially available glycoprotein that has twoN-linked biantennary complex glycans (36). While none of the rPA-ILNmproteins were detected binding to transferrin, they were all found tobind to asialotransferrin (FIG. 2A). This demonstrated that, unlike theparental rPA-ILN protein, the selected rPA-ILNm proteins exhibitedspecificity for LacNAc, which displays terminal β1,4-linked galactose,exposed on the antenna of N-linked glycans as a result of the removal ofterminal sialic acid. Removal of the terminal β1,4-linked galactosethrough treatment with β1,4-galactosidase, generatingagalactotransferrin, eliminated binding of the rPA-ILNm proteins (FIG.2A). This confirmed that binding was dependent on the terminalβ1,4-linked galactose. A similar response pattern was observed forfetuin, asialofetuin and agalactofetuin glycoforms (FIG. 2B). Theresidual signals observed against agalactofetuin, generated by treatmentof asialofetuin with β1,4-galactosidase, could have been due to bindingto terminal β1,3-linked galactose displayed by de-sialylated O-linkedThomsen-Friedenreich (T) antigen structures on this glycoprotein (36).However, the plant lectin ECL was also found to generate residualsignals against agalactofetuin. This lectin is known to be specific forterminal β1,4-linked galactose and does not bind significantly toterminal β1,3-linked galactose (38). This suggested incomplete removalof terminal β1,4-linked galactose was the likely source of the residualbinding signals.

Identification of Amino Acid Substitutions in Selected rPA-ILNmProteins—Plasmid DNA from each of the rPA-ILNm expressing clones wasisolated and sequenced to determine the nature of the aminosubstitutions present in each protein (FIG. 3). It can be clearly seenthat the functional ELLA screening process resulted in theidentification of a collection of rPA-ILNm proteins in which specificamino acid substitutions occurred with high frequency at each of themutagenized positions. Of the nine rPA-ILNm proteins isolated, threewere found to have a H50V substitution and two had a H50N substitution.Examining the Asp52 position, it can be seen that three rPA-ILNmproteins were found to have a D52C substitution while another three hada D52N substitution. Also, arginine was found to be substituted forGln53 in four rPA-ILNm proteins. We undertook more detailed analysis ofthis collection of rPA-ILNm proteins to ascertain the potentialsignificance of these amino acid substitutions

Lectin Dose Response Curves Against Defined Glycoconjugate Targets—Thespecificity of the selected rPA-ILNm proteins was further assessed bygenerating lectin dose response curves against two specificglycoconjugate targets; BSA-LacNAc and BSA-αGal (FIGS. 4A & B). Theseglycoconjugates enable detection of potentially weak interactions (39),due to multivalent and high density display of glycans, and assessmentof the impact of substitutions on carbohydrate binding selectivity i.e.binding to glycans with terminal α-linked versus β-linked galactose(FIG. 4C). As all of the lectins molecules to be assessed had anequivalent quaternary structure, these lectin dose response curvesenabled comparative analysis of the relative affinities of each of therPA-ILNm lectins for each of the glycoconjugates.

The parental rPA-ILN protein only showed very weak binding to theBSA-LacNAc glycoconjugate, even at relatively high lectin concentrationsof 10 μg mL⁻¹ (not shown in FIG. 4A). This was expected since the PA-ILprotein is known not to bind significantly to glycans with terminalβ1,4-linked galactose (23,28). Conversely, all of the rPA-ILNm proteins,with the exception of the rPA-ILNmA8, displayed strong binding to theBSA-LacNAc glycoconjugate. Of particular note was the rPA-ILNmE6protein, which displayed a very high relative affinity to BSA-LacNAc,indicated by a very rapid increase in signal strength with increasinglectin concentration, and signals ultimately reached saturation at alectin concentration of 0.625 μg mL⁻¹. The rPA-ILNmB10 and rPA-ILNmF3proteins also showed a high relative affinity for the BSA-LacNAcfollowed by rPA-ILNmC5>G3-F6>B4. The parental rPA-ILN protein wasobserved to bind to the BSA-αGal glycoconjugate with a higher relativeaffinity than any of the rPA-ILNm proteins (FIG. 4B). However, four ofthe rPA-ILNm proteins also displayed a capacity to bind strongly to theBSA-αGal glycoconjugate. Of these, the rPA-ILNmF3 and rPA-ILNmB10proteins displayed the highest relative affinity to the BSA-αGalglycoconjugate, exhibiting similar dose response curves, while therPA-ILNmB4 protein displayed a lower binding capability. The rPA-ILNmE6protein also showed some capacity to bind the BSA-αGal conjugate butonly at relatively high concentrations of the lectin and signalstrengths did not reach saturation over the lectin concentration rangeexamined. The remaining rPA-ILNm proteins, in particular rPA-ILNmC5 andrPA-ILNmF6 proteins, showed very little binding to BSA-αGal even atrelatively high lectin concentrations of 10 μg mL⁻¹ (data not shown).The data against the two glycoconjugates clearly showed that while someof the rPA-ILNm proteins, like rPA-ILNmE6 and rPA-ILNmC5, exhibitedselectivity towards BSA-LacNAc; others, like rPA-ILNmF3, exhibited dualbinding specificities for α- and β-linked galactose by binding bothglycoconjugates (FIG. 4C).

Lectin Dose Response Curves on Natural Glycoproteins Glycoforms—Lectindose response curves were also generated against asialotransferrin andasialofetuin (FIGS. 5A & 5B). These natural glycoproteins displayglycans at significantly lower densities than the glycoconjugates. Dataobtained using these targets therefore gives a greater, and morebiologically relevant, insight into the binding activities anddependencies of the rPA-ILNm proteins. The rPA-ILNmE6 protein displayeda very high relative affinity for both asialotransferrin andasialofetuin with lectin dose response curves reaching saturation forboth glycoproteins at lectin concentrations above 1 μg⁻¹. The rPA-ILNmF3and rPA-ILNmB10 proteins also displayed high relative affinities to bothasialotransferrin and asialofetuin, albeit not as high as rPA-ILNmE6.While the rPA-ILNmC5 was observed to bind strongly to asialofetuin, itonly bound relatively weakly to asialotransferrin. This indicated thatbinding of this protein to LacNAc is potentially more dependent on thedensity or distribution of glycans, and possibly more reliant on avidbinding, than rPA-ILNmE6. The rPA-ILNmG3 protein showed similarresponses to both glycoproteins as rPA-ILNmC5. The rPA-ILNmF6 proteindisplayed a higher relative affinity for both asialotransferrin andasialofetuin than either the rPA-ILNmC5 or rPA-ILNmG3 proteins andgenerated a lectin dose response curve against asialofetuin comparableto that of the rPA-ILNmF3 and rPA-ILNmB10 proteins. This indicates thatthe rPA-ILNmF6 is potentially less dependent on high density display oftarget ligands for effective binding and has a higher affinity forterminal β-linked galactose than rPA-ILNmC5 or rPA-ILNmG3.

Determination of Affinity Constants for rPA-ILNm Proteins—A relativeaffinity constant, K_(D), was calculated for selected rPA-ILNm proteinsagainst the BSA-LacNAc glycoconjugate according the method described byKirkeby et al 2002. K_(D) is defined as being the concentration of theglycoconjugate required to fill half of the available lectin bindingsites at equilibrium. If a lectin has a high affinity for theglycoconjugate, then the K_(D) will be low as it will take a lowerconcentration of glycoconjugate to bind half of the lectin molecules.The calculated K_(D) for rPA-ILNmE6, rPA-ILNmF3 and ECL for BSA-LacNAcwere 4 ng, 6.3 ng and 21 ng, respectively (FIG. 6) indicating that therPA-ILNmE6 protein had approximately a 5 fold greater affinity for theBSA-LacNAc glycoconjugate than ECL while rPA-ILNmF3 had a 3 fold greateraffinity.

Application of Immobilized rPA-ILNmE6 for Glycoprotein and GlycoformIsolation—to evaluate the ability of rPA-ILNmE6 to be used for selectiveglycoprotein and glycoform isolation and purification. The lectin wasimmobilized onto CNBr activated Sepharose 4B. The lectin readilyimmobilized at high densities and lectin immobilization densities ofapproximately 20 mg mL⁻¹ of resin were reproducibly achieved. Toevaluate the ability of this lectin affinity resin to isolateglycoproteins displaying terminal LacNAc, we first performed simplelectin pull down assays in eppendorf tubes using a test protein mixtureprepared by mixing asialotransferrin, glucose oxidase (displays highmannose), cytochrome C and carbonic anhydrase (both non-glycosylated).Fractions of unbound and bound protein were ultimately evaluated bySDS-PAGE (FIG. 7A). As can be seen from FIG. 7A, the rPA-ILNmE6Sepharose resin selectively extracted the asialotransferrin from theprotein mixture and the protein was effectively recovered by theincorporation of free galactose into elution buffers. The rPA-ILNmE6Sepharose resin was also packed into 1 mL FPLC column housings to enableeasy attachment to FPLC systems. We evaluated the ability of this columnto efficiently separate glycoforms of transferrin. A test samplecontaining equal amounts of transferrin and asialotransferrin wasinjected onto the FPLC column and was separated into two clearfractions, one unbound protein fraction and one galactose eluted boundprotein fraction (FIG. 7B). Both the bound and unbound fractions wereassessed by ELLA to evaluate their glycoprotein composition (FIG. 7C).In ELLA's transferrin was shown to generate strong responses from thesialic acid specific lectin SNA but did not respond significantly witheither of the galactophilic lectins ECL or rPA-ILNmE6. Asialotransferrinshowed a reduced response to the SNA but significantly increasedresponses to both of the galactophilic lectins. While this confirmed thepresence of terminal β1,4-linked galactose and it also indicated thatthe neuraminidase treatment used in the preparation of theasialotransferrin had in fact generated partially desialylatedglycoforms. When the FPLC fractionated material was assessed only thebound fraction was found to elicit responses from the galactophiliclectins. This indicated that the rPA-ILNmE6 Sepharose column hadefficiently separated the transferrin and asialotransferrin glycoformsinto two distinct populations and that it had effectively isolated thepartially desialylated transferrin glycoforms.

DISCUSSION

In the present study, we have demonstrated how the carbohydrate bindingspecificity of the α-galactophilic PA-IL protein could be significantlyaltered through random mutagenesis of specific amino acid residues inits binding site. We identified a number of novel RPL's exhibitingspecificity and high affinity for glycoproteins displaying LacNAc and anaffinity for this glycan epitope significantly greater than that ofcommercially available plant lectin ECL (FIG. 6). While some of therPA-ILNm proteins displayed distinct selectivity towards LacNAc(rPA-ILNmE6 and rPA-ILNmC5), others displayed dual specificity bindingto both LacNAc and glycans with terminal α-linked galactose (rPA-ILNmF3and PA-ILNmB10) (FIG. 4C). In addition to this, there were cleardifferences in the relative affinities of rPA-ILNm proteins fordifferent glycoprotein targets. Through the functional characterisationof this collection of mutants, and identification of the specific aminoacid substitutions in each, we were able to identify specificsubstitutions at each of the mutagenized positions in the parentalrPA-ILN protein that were linked with specific carbohydrate bindingactivities.

The Role of Specific Amino Acid Substitutions in Dictating CarbohydrateBinding Properties—Of the rPA-ILNm proteins identified, the rPA-ILNmE6protein appeared to exhibit a very high relative affinity forBSA-LacNAc. While this protein also showed some capacity to bind to theBSA-αGal glycoconjugate, it only did so relatively weakly when comparedto its response to the BSA-LacNAc glycoconjugate (FIG. 4C). TherPA-ILNmC5 also appeared to bind well to BSA-LacNAc, albeit not as wellas rPA-ILNmE6 (FIG. 4B), but was not observed to bind to the BSA-αGalconjugate (FIG. 4A). When these two proteins were examined againstasialofetuin and asialotransferrin, it became clear that the rPA-ILNmE6potentially had a significantly higher affinity for LacNAc than therPA-ILNmC5 protein, which appeared to be more dependant of the densityof glycan display (FIG. 5). The rPA-ILNmC5 protein carries the same H50Nsubstitution present in rPA-ILNmE6 but carries different substitutionsat the Asp52 and Gln53 positions. These results indicate that, while theH50N substitution may be associated with high affinity binding toLacNAc, the D52N and Q53G substitutions present in rPA-ILNmE6 play arole in further modulating its carbohydrate binding properties resultingin its significantly higher relative binding affinities for LacNAccompared to rPA-ILNmC5.

The responses of another group of rPA-ILNm proteins indicated that aH50V substitution could also support high affinity binding to LacNAc.This substitution was present in rPA-ILNmF3, rPA-ILNmB10 and rPA-ILNmG3and all of these proteins were observed to bind strongly to theBSA-LacNAc glycoconjugate. Analysis against asialofetuin andasialotransferrin again indicated however that the rPA-ILNmB10 andrPA-ILNmF3 proteins displayed a higher relative affinity for LacNAc thanthe rPA-ILNmG3, although not as high as that displayed by the rPA-ILNmE6protein. The rPA-ILNmF3 and rPA-ILNmB10 proteins also displayed strongbinding to the BSA-αGal, albeit not as strong as the parental rPA-ILNprotein, and this was not observed for the rPA-ILNmG3 protein. As thesethree proteins only differ from each other by possessing differentsubstitutions at positions 52 and 53, this again demonstrates that aminoacids at these positions play a role in further defining the specificityand affinity of the rPA-ILNm proteins. The rPA-ILNmB10 and rPA-ILNmF3proteins actually only differ from each other at one position, carryinga Q53E and a Q53R substitution respectively. The Q53R substitution wasalso present in rPA-ILNmB4 which also binds well to the BSA-αGal. Thesedata indicated that substitution of Gln53 with a basic (Arg) or acidic(Glu) residue could be linked with a higher affinity for α-linkedgalactose.

Another interesting observation was that, while the lectin dose responsecurves for rPA-ILNmF3 and rPA-ILNmB10 proteins on asialotransferrin didnot increase as rapidly as that of rPA-ILNmE6, indicative of a lowerrelative affinity, they ultimately reached a higher absorbance plateauindicating a greater final density of these proteins bound to thesurface. However, we had observed that both of these proteins had atendency to form aggregates when high protein concentration stocksolutions, stored at −80° C., were being defrosted. These aggregatesgenerally went back into solution when samples were fully thawed but allsamples were centrifuged to ensure removal of any residual proteinaggregates prior to use. These proteins occasionally also generated highsignals in negative control wells. This was also observed for therPA-ILNmE12 protein, albeit more consistently, leading to it beingexcluded from further analysis. The one common feature of all three ofthese proteins was the occurrence of a D52C substitution and it ispossible that this residue could mediate protein aggregation at highlectin concentrations through disulfide bond formation. As a result, thehigher saturation signals obtained for these lectins onasialotransferrin may be the result of binding of protein aggregatesformed at high lectin concentrations.

Novel Glycoanalytical Tools for Applications in the LifeSciences—Lectins have found widespread applications within the field ofglycobiology and have been implemented in a diverse range of formats tocharacterise the glycosylation status, and to detect changes inglycosylation, of biomolecules. Changes in the glycosylation of proteinsor cell surfaces can be concurrent with, and indicative of, a change inthe physiological status of a cell or the development of a disease stateand can therefore be used as a means of diagnosis (1-5). LacNAc is animportant glycan epitope commonly displayed on cell surfaces and as partof the antenna of complex N-linked glycan structures of glycoproteins.For example, serum IgG's, unlike many other serum glycoproteins, are notheavily sialylated and the N-linked glycans present in the Fc region ofthe glycoproteins usually bear biantennary glycans terminating in LacNAc(11,24). A reduction in terminal β1,4 galactosylation of these N-linkedglycans has been diagnostically linked with a number of autoimmunedisorders including rheumatoid arthritis while increased galactosylationindicates remission of the disease (11-14,40). The RPL's we havedeveloped were clearly demonstrated to be capable of sensitivelydetecting glycoproteins displaying terminal LacNAc and of being capableof differentiating between different glycoprotein glycoforms (FIG. 2).Lectin affinity chromatography (LAC) is also widely used for theseparation and isolation of glycoproteins. With more than 50% ofproteins being glycosylated LAC is a particularly powerful tool forglycoproteomic analysis. Proteomic samples are highly complex andglycoproteins in biological materials are often only present in verysmall quantities and efficient isolation and pre-concentration of thesemolecules is essential for their identification and characterisation.LAC is often also used as an initial step to pre-concentrateoligosaccharides, glycopeptides, or to separate glycoforms, prior to MSbased glycoanalysis (39,41-43). We clearly demonstrated that the novelgalactophilic RPL's reported here could be immobilized at high densitiesonto solid support matrices, such as Sepharose, to generate highlyeffective bioaffinity matrices enabling efficient separation andselective purifification of glycoproteins and glycoforms displayingterminal β1,4-linked galactose (FIG. 7). The RPL's reported here couldtherefore find widespread applications in the fields of functionalglycomics and proteomics.

Many biopharmaceutical products are glycosylated molecules andvariations in glycosylation of bio-therapeutics can have a verysignificant impact on a products physiochemical properties, efficacy,and immunogenicity (11,44-47). Sialylation of some bio-therapeutics,such as Erythropoietin (EPO), can have a significant impact on theirphysiochemical properties, blood retention and overall efficacy(44,46,48,49). Monitoring of sialylation of these products is often animportant determinant in the production of these products and methodsusing lectins, such as ECL, to monitor for changes in sialylation havebeen reported in the literature (48,50). Monoclonal antibodies (MAb's)represent a very significant and rapidly growing class ofbiotherapeutics (11,24). The N-linked glycans in the Fc region of MAb'sare usually terminated in galactose and these glycans are essential forthe ability of MAb's to elicit ADCC (Antibody Dependent CellularCytotoxicity) and CDC (Complement Dependent Cytotoxicity) effectorfunctions vital for their efficacy (11,12,24,40,51). As with theanalysis of other glycoproteins, LAC can enable more efficient analysisand characterisation of glycosylated biotherapeutics. With theirspecificity for LacNAc, the RPL's reported here could be particularlyuseful in the analysis of MAb's to determine the extent of terminalgalactosylation which is often a major source of heterogeneity in theseproducts (12). In addition to the many potential analytical scaleapplications, the ability to readily scale the production of our novelRPL's, could also enable them to ultimately overcome the many barriersthat have limited the application of other eukaryotic lectins and enablethem to be applied at a production scale, in a way analogous to ProteinA, for the selective purification of optimal biotherapeutic glycoformsto produce safer more efficacious drugs.

EXAMPLE B Experimental Procedures

Site Directed Mutagenesis—PCR based site directed mutagenesis of thelecA gene, which encodes the PA-IL protein, was achieved as described inExample A. The pQE30PA-IL vector is an Escherichia coli expressionvector which expresses the rPA-ILN protein (recombinant PA-IL proteinwith an N-terminally positioned hexa-histidine (6HIS) affinitypurification tag) and this was used as a template for whole vectoramplification (See Example A—Table A1). Whole vector amplification wasachieved using 5′ phosphorylated primers designed to anneal within thelecA sequence with their 5′ ends exactly next to each other. The reverseprimers were designed to overlap the region to be mutagenized. Thisenabled the introduction of mutations through manipulation of reverseprimer sequences while the sequence of the forward primer, PA-ILmutF,was kept constant (Table B2). Successful PCR reactions were purified andsubjected to digestion with the restriction enzyme DpnI to selectivelydigest the parental template vector DNA. Digestions were ultimately runon agarose gels and PCR products corresponding to the expected size oflinear vector were gel extracted. The final purified PCR products, withblunt phosphorylated ends, were re-circularised by simple self-ligationand the ligated DNA transformed into E. coli strain KRX. Typically threetransformants were picked into overnight 10 mL Terrific Broth (TB)cultures supplemented with 50 μM IPTG and expression of mutant rPA-ILNproteins (rPA-ILNm) confirmed by SDS-PAGE analysis of total cellularprotein. Plasmid DNA was isolated from clones expressing proteins of theexpected size and successful introduction of the desired mutationsconfirmed by DNA sequencing (MWG-Eurofins). All of the plasmids used inthis study are described in Table B1 and all of the primers used aredescribed in Table B2.

TABLE B1 Plasmids Constructed in Example B. Plasmids EncodingMutagenised rPA-ILN (rPA-ILNm) Proteins Protein Amino Acid Plasmid NameExpressed Substitutions Source H50 Single Mutants pPC30PA-IL-A H50A H50AExample B pPC30PA-IL-V H50V H50V Example B pPC30PA-IL-L H50L H50LExample B pPC30PA-IL-F H50F H50F Example B pPC30PA-IL-P H50P H50PExample B pPC30PA-IL-S H50S H50S Example B pPC30PA-IL-T H50T H50TExample B pPC30PA-IL-N H50N H50N Example B pPC30PA-IL-Q H50Q H50QExample B pPC30PA-IL-D H50D H50D Example B pPC30PA-IL-E H50E H50EExample B pPC30PA-IL-K H50K H50K Example B pPC30PA-IL-R H50R H50RExample B H50N:Q53 Double Mutants pPC30PA-IL-NA H50N:Q53A H50N:Q53AExample B pPC30PA-IL-NV H50N:Q53V H50N:Q53V Example B pPC30PA-IL-NLH50N:Q53L H50N:Q53L Example B pPC30PA-IL-NG H50N:Q53G H50N:Q53G ExampleB pPC30PA-IL-NS H50N:Q53S H50N:Q53S Example B pPC30PA-IL-NY H50N:Q53YH50N:Q53Y Example B pPC30PA-IL-NN H50N:Q53N H50N:Q53N Example BpPC30PA-IL-ND H50N:Q53D H50N:Q53D Example B pPC30PA-IL-NE H50N:Q53EH50N:Q53E Example B pPC30PA-IL-NK H50N:Q53K H50N:Q53K Example BpPC30PA-IL-NR H50N:Q53R H50N:Q53R Example B pPC30PA-IL-NH H50N:Q53HH50N:Q53H Example B Additional H50:Q53 Double Mutants pPC30PA-IL-VKH50V:Q53K H50V:Q53K Example B pPC30PA-IL-VR H50V:Q53R H50V:Q53R ExampleB pPC30PA-IL-QK H50Q:Q53K H50Q:Q53K Example B pPC30PA-IL-QR H50Q:Q53RH50Q:Q53R Example B Q53 Single Mutants pPC30PA-IL-HK Q53K Q53K Example BpPC30PA-IL-HR Q53R Q53R Example B pPC30PA-IL-HE Q53E Q53E Example B

TABLE B2Primer Sequences Used in Example B for Site Directed Mutagenesis of rPA-ILN.Forward Primer PA-ILmutFCgttttgtggtgcgctggtcatgaagattggc - SEQ ID NO. 48Reverse Primers Used for Generation of Specific H51 Mutants H50A

H50V

H50L

H50F

H50P

H50S

H50T

H50N

H50Q

H50D

H50E

H50K

H50R

Reverse Primers Used for Generation of Specific Double Mutants H50N:Q53A

H50N:Q53V

H50N:Q53L

H50N:Q53G

H50N:Q53S

H50N:Q53Y

H50N:Q53N

H50N:Q53D

H50N:Q53E

H50N:Q53K

H50N:Q53R

H50N:Q53H

H50V:Q53K

H50V:Q53R

H50Q:Q53K

H50Q:Q53R

Reverse Primers Used for Generation of Specific Q53 Mutants Q53K

Q53R

Q53E

Protein Expression and Purification—For protein expression, plasmidswere transformed into the protease deficient E. coli strain KRX (30).Expression clones were cultured in Terrific Broth (TB). Cultures weregrown at 37° C. with shaking at 200 rpm until an optical density of 0.6at 600 nm was reached and then induced by addition of IPTG to a finalconcentration of 50 μM. Cultures were then placed at 30° C. with shakingat 200 rpm for overnight incubation. Cells were harvested bycentrifugation and cell pellets resuspended in lysis buffer (10 mMNaH₂PO₄, 300 mM NaCl, 40 mM imidazole, pH 8.0). Cell disruption wasachieved by high pressure using a Constant Systems cell disrupter andcell debris was removed by centrifugation. Clarified cell lysates wereapplied to 10 mL IMAC columns (IMAC Hypercel from Pal) and a highstringency wash buffer with 100 mM imidazole was used to removenon-specifically bound contaminating proteins. The desired 6HIS taggedproteins were ultimately eluted using 250 mM immidizole and elutedproteins were aliquoted and stored at −80° C. in the elution buffer.Typical yields were around 200 mg per 250 mL starting culture. Purifiedproteins were analysed by SDS-PAGE to assess purity and routinely bufferexchanged and concentrated using Vivaspin centrifugal membrane devices(Sartorius-Stedim), with a molecular weight cut off of 10 kDa, accordingto the manufacturer's guidelines.

General Enzyme Linked Lectin Assay (ELLA) Method—The Gal-α1,3-Gal-BSA(BSA-αGal) and Gal-β1,4-GlcNAc-BSA (BSA-LacNAc) glycoconjugates usedwere from Dextra Laboratories and presented on average 20 glycanmoieties per BSA molecule. Biotinylated plant lectins GSL-I (Griffoniasimplicifolia isolectin B4), ECL (Erythrina cristagalli Lectin), RCA(Ricinus communis Agglutinin), SNA (Sambucus nigra Agglutinin) and MALII(Maackia amurensis Lectin) were from Vector Laboratories. Humantransferrin was from Sigma Aldrich and asialotransferrin (AsT) wasgenerated by treatment using neuraminidase (Clostridium perfingens) inaccordance with manufacturer's guidelines (New England Biolabs). ELLA'swere essentially performed according to the method described by Thompsonet al (2011) (33). More specifically, glycoproteins were prepared in PBSand typically immobilized at a concentration of 5 μg mL⁻¹. Forqualitative ELLA's lectins were assayed at a concentration of 2 μg mL⁻¹in TBST (20 mM Tris, 150 mM NaCl, 0.05% Tween-20, 1 mM CaCl₂, 1 mMMnCl₂, 1 mM MgCl₂, pH 7.6). For lectin dose response experiments, eachlectin was evaluated at a range of concentrations prepared by serial 1:2dilution of an initial lectin solution of 2 μg mL⁻¹ to a finalconcentration of 31 ng mL⁻¹. Binding of 6HIS tagged rPA-IL proteins wasdetected after 1 hour incubation at 25° C. using a HRP conjugatedanti-HIS antibody diluted 1:10,000 in TBST (Sigma Aldrich). Biotinylatedplant lectins were detected using a HRP conjugated anti-biotin antibodydiluted 1:10,000 in TBST (Sigma Aldrich).

Protein Structural Modelling and Image Rendering—In silico analysis ofthe PA-IL protein and its carbohydrate binding site was carried outusing the PDB file 2VXJ (23). Structural models were generated usingDeep View (Swiss Model) (25) and models generated were ultimatelyrendered using the CCP4MG software (26).

RESULTS

In Example A, we constructed an expression vector, pQE30PA-IL, enablingthe expression of rPA-IL with an N-terminally positioned poly-histidinetag (rPA-ILN). This poly-histidine (6HIS) tag enabled rapid and simplepurification of the rPA-ILN protein by IMAC and therefore independentlyof its carbohydrate binding specificity. Positioning of thepoly-histidine tag at the N-terminus of the rPA-IL protein was found todisrupt the quaternary structure of the protein resulting in theformation of homodimers rather than the native tetrameric configurationadopted by the wild type untagged PA-IL protein. Despite this, therPA-ILN protein, and derived rPA-ILNm proteins, were demonstrated inExample A to be active and binding could be detected with highsensitivity in ELLA's against defined glycoprotein targets. ThepQE30PA-IL vector was therefore selected as the target DNA molecule formutagenesis studies undertaken in this work. As all of the rPA-ILNmgenerated would exhibit an equivalent quaternary structure to theparental rPA-ILN protein, comparative analysis of carbohydrate bindingspecificity and affinity could be performed by ELLA to assess the impactof specific amino acid substitutions.

The His50 residue is critical for dictating the binding specificity ofrPA-ILN—Work conducted in Example A indicated that the His50 residue inthe binding pocket of the PA-IL protein played a critical role indetermining the carbohydrate binding specificity of the protein. Toindependently examine the role of this amino acid residue, it wassubstituted with 13 alternative amino acids selected to berepresentative of the full spectrum of potential amino acid prosperities(Table B1 above). All of the resulting rPA-ILNm proteins weresuccessfully expressed in E. coli and purified by IMAC with theexception of one carrying a H50D mutation, which was found to beinsoluble. An rPA-ILNm protein carrying a H50P substitution was observedto express weakly in E. coli and, when purified protein was assessed bySDS-PAGE, it generated multiple high molecular weight bands indicatingthat it potentially formed aggregates (data not shown). The carbohydratebinding properties of each of the remaining 12 successfully purifiedHis50 substituted rPA-ILNm proteins was qualitatively assessed byperforming ELLA's against two specific BSA glycoconjugate targets;Gal-α1,3-Gal-BSA (BSA-αGal) and Gal-β1,4-GlcNAc-BSA (BSA-LacNAc) (FIG.13). The glycoprotein asialotransferrin (AsT) was also included in thisinitial screen. This glycoprotein has only two N-linked glycans (36) andtherefore displays a significantly lower density of glycans withterminal LacNAc than the BSA-LacNAc glycoconjugate. We had observed inExample A that, while BSA-LacNAc was useful for detecting weak bindingevents, AsT was more useful in detecting subtle differences in therelative binding affinities of proteins where binding might be moredependent on the surface distribution and/or density of glycan moieties.Several of the random mutants generated in Example A (Table A1 above),and a number of commercially available plant lectins, were also includedin these qualitative assays for comparative purposes. These screensidentified a number of amino acid substitutions that significantlyaltered the carbohydrate binding properties of the parental rPA-ILNprotein, including some that generated rPA-ILNm proteins exhibitingsignificant binding to LacNAc (FIG. 13). This clearly demonstrated thatsubstitution of the His50 residue alone was sufficient to altercarbohydrate binding properties.

Specific His50 substitutions result in high affinity binding to terminalβ1,4-linked galactose Initial screens identified a number of His50substitutions that generated rPA-ILNm proteins exhibiting strong bindingto BSA-LacNAc (FIG. 13). Interestingly, these included H50N, H50V andH50T substitutions that had been observed in randomly mutated rPA-ILNmproteins identified in Example A as exhibiting high affinity forglycoproteins displaying terminal β1,4-linked galactose (Table A1). Inaddition to these substitutions, H50E and H50Q substitutions were alsoobserved to generate proteins exhibiting strong binding to BSA-LacNAc.While an rPA-ILNm protein carrying a H50P substitution was observedbinding to BSA-LacNAc, it was also observed to form aggregates when runon SDS-PAGE. The true affinity of this protein for LacNAc wouldtherefore be difficult to interpret and to compare to that of the otherrPA-ILNm proteins, so further analysis of this protein was notconducted. A protein carrying a H50L substitution was also observed tobind to BSA-LacNAc but the signal obtained was weaker than that observedfor the H50V or H50T proteins. Lectin dose response curves weregenerated for each of the five His50 substituted rPA-ILNm proteins thatgenerated the strongest signals against BSA-LacNAc in the initial ELLAscreens (FIG. 14A). This enabled a more quantitative comparison of therelative affinities of these proteins for the glycoconjugate and a morequantitative assessment of the impact of each of the specific His50substitutions on the carbohydrate binding properties of the rPA-ILNprotein.

Of all of the His50 substitutions made, the H50N protein exhibited thehighest relative affinity for BSA-LacNAc. This was only slightly weakerthan that observed for the random mutant rPA-ILNmE6 protein whichcarries the same H50N substitution but also carries two additional D52Nand Q53G amino acid substitutions (FIG. 14A). However, the H50Ngenerated significantly lower signals on AsT, approximately half thatobserved for the rPA-ILNmE6 protein (FIG. 13), indicating that it had asignificantly lower relative affinity for glycans with terminal LacNAcand that binding was more dependent on the surface density and thecontext in which glycans were displayed. This behaviour was alsoobserved for another random mutant, rPA-ILNmC5, which also carried aH50N substitution along with D52T and Q53S substitutions (FIG. 13). TheH50E protein was also observed to bind strongly to BSA-LacNAc displayinga relative binding affinity that was only slightly weaker than thatobserved for the H50N protein (FIG. 14A). However, it failed to bindsignificantly to AsT (FIG. 13) indicating that it potentially had aneven lower relative affinity for LacNAc, and an even greater dependencyon the density of glycan display, than the H50N protein. The H50Vprotein generated binding signals against BSA-LacNAc comparable to thatof the random mutant, rPA-ILNmF3, which carries the same H50Vsubstitution along with D52C and Q53R substitutions. However, incontrast to the rPA-ILNmF3 protein, H50V failed to bind significantly toAsT, indicating it had a lower relative higher affinity for glycans withterminal β1,4-linked galactose (FIG. 13). Lectin dose response curvesdemonstrated that the relative binding affinity of H50V for BSA-LacNAcwas significantly lower than that observed for either the H50N or theH50E proteins but greater than that observed for the H50Q and H50Tproteins (FIG. 14A).

His50 substitutions result in reduced binding affinities for α-linkedgalactose—All of the His50 substitutions negatively impacted on theability of rPA-ILNm proteins to bind to the BSA-αGal glycoconjugate.Proteins carrying either a H50Q or a H50K substitution exhibited thestrongest binding to the BSA-αGal glycoconjugate (FIG. 13) but theirrelative affinities for this conjugate were still significantly lowerthan that of the parental rPA-ILN protein (FIG. 14B). The H50K protein,like the parental rPA-ILN protein, bound relatively strongly to BSA-αGalbut did not bind to BSA-LacNAc (FIG. 13). The H50Q protein, however,exhibited dual specificity exhibiting relatively strong binding signalson BSA-LacNAc and the strongest binding of all of the His50 mutantstoward the BSA-αGal glycoconjugate (FIG. 13).

The H50N protein generated significant binding signals against BSA-αGal(FIG. 13) and lectin dose response curves showed that it had asignificantly higher affinity for this glycoconjugate than that observedfor the rPA-ILNmE6 protein (FIG. 14B). Therefore, while this proteinstill exhibited significant preferential binding to BSA-LacNAc, it wasnot as selective as the rPA-ILNmE6 protein. While the H50V protein hadbeen observed to bind with relatively high affinity to BSA-LacNAc, itdid not exhibit significant binding to the BSA-αGal glycoconjugate. Thiswas in contrast to the rPA-ILNmF3 protein that was observed to bindrelatively strongly to both BSA glycoconjugates (FIG. 13). Similarly,the H50T protein bound to BSA-LacNAc with comparable strength to thatobserved for the rPA-ILNmB4 protein but, unlike the rPA-ILNmB4 protein,it exhibited negligible binding to the BSA-αGal glycoconjugate (FIG.13).

The Q53 and D52 residues play a role in modulating carbohydrate bindingspecificity and Affinity—Analysis of the rPA-ILNm proteins with singleHis50 substitutions, and comparison with the sugar binding properties ofclosely related randomly mutated rPA-ILN proteins, clearly indicatedthat the Gln53 and D52 residues played a role in further modulating thecarbohydrate binding properties of the rPA-ILNm proteins. We firstexamined the impact of Gln53 substitutions when made in conjunction witha H50N substitution. The plasmid encoding the H50N protein wasmutagenized using primers specifically designed to introduce anadditional specific amino acid substitution in place of the Gln53residue (Table B2 above). This generated twelve new expression vectors,each expressing an rPA-ILNm protein with the H50N substitution incombination with one of 12 different amino acid substitutions in placeof the Gln53 residue (Table B1). One of the resulting proteins,H50N:Q53Y, was found to be insoluble and was not characterised further.The remaining 11 mutants were evaluated as before by performing ELLAanalysis against BSA-LacNAc, BSA-αGal and AsT (FIG. 15). All of theH50N:Q53 double mutants exhibited comparable activity when testedagainst BSA-LacNAc. Examination of binding signals towards AsT revealedsubtle differences in binding affinity with most substitutions resultingin a slight reduction in binding signals. This was particularlyunexpected in the case of the H50N:Q53G double substitution. Based onthe carbohydrate binding properties of the rPA-ILNmE6 protein, one mighthave expected that the introduction of a Q53G substitution would resultin some enhancement in binding to AsT and a closer approximate activityto that of the rPA-ILNmE6 protein. Instead binding was actually weakerthan that of the H50N protein and was less than half the strength of thesignal observed for the rPA-ILNmE6 protein. Most Gln53 substitutionsalso resulted in a significant reduction in binding to the BSA-αGalglycoconjugate compared to the parental H50N protein. While binding tothe BSA-αGal glycoconjugate was generally reduced to levels comparableto that of the rPA-ILNmE6 protein, most of the proteins also bound moreweakly to AsT suggesting that this apparent increase in selectivity wasin fact due to an overall reduction in the affinity of carbohydratebinding. However, one protein of note was the H50N:Q53E protein whichdisplayed significantly reduced binding to AsT compared to the parentalH50N but displayed comparable, and potentially slightly stronger,binding to the BSA-αGal. This suggests that the Q53E substitution mightpreferentially promote binding to terminal α-linked galactose overβ-linked galactose.

Q53R substitutions promote binding to α-linked galactose—Comparison ofthe binding specificities of the H50T and H50V proteins with that of therPA-ILNmB4 and rPA-ILNmF3 proteins respectively, suggested that a Q53Rsubstitution might promote binding to terminal α-linked galactose. Wetherefore introduced a Q53R substitution into the H50V protein and theresulting H50V:Q53R protein was observed to bind more strongly to theBSA-αGal glycoconjugate (FIG. 16A). Lectin dose response curves showedthat this double mutant exhibited a comparable relative affinity forBSA-αGal to that of the rPA-ILNmF3 protein (FIG. 16B). The H50V:Q53Rprotein also showed significantly stronger binding to AsT than theparental H50V and this was also comparable to that observed for therPA-ILNmF3 protein (FIG. 16A). Substitution of the Gln53 residue with alarger lysine residue generated the H50V:Q53K protein which displayed anoverall reduced carbohydrate binding activity compared to the parentalH50V protein (FIG. 16A). We introduced these same Gln53 substitutionsinto the H50Q protein to see if there would be a similar impact oncarbohydrate binding properties. While the H50Q:Q53R protein wasobserved to bind with a slightly higher relative affinity to theBSA-αGal glycoconjugate compared to the parental H50Q protein (FIG.16B), binding to the BSA-LacNAc glycoconjugate was abolished (FIG. 16A).

The above results suggested that a Q53R substitution could promotebinding to glycans with terminal α-linked galactose. However, neitherthe H50Q:Q53R protein nor the H50V:Q53R proteins bound to the BSA-αGalglycoconjugate as strongly as the rPA-ILN protein due to substitution ofthe His50 residue (FIG. 16B). We therefore introduced the Q53Rsubstitution into the original rPA-ILN protein to determine if it wouldresult in a protein with enhanced affinity for the BSA-αGalglycoconjugate. As expected, the resulting Q53R protein was observed todisplay a higher relative affinity for the BSA-αGal than the parentalrPA-ILN protein and it did not bind to BSA-LacNAc (FIGS. 16A & B).Introduction of a Q53K substitution resulted in almost a completeabolition of binding to either the BSA-LacNAc or BSA-αGalglycoconjugates (FIG. 16A). Based on earlier results observed for aH50N:Q53E double mutant, we also evaluated the impact of introducing aconservative Q53E substitution into the parental rPA-ILN protein. Theresulting Q53E protein was found to exhibit an even further enhancedrelative affinity for BSA-αGal then the previously made Q53R protein(FIG. 16B).

DISCUSSION

In Example B, we set out to independently assess the roles of the His50,Asp52 and Gln53 residues in the carbohydrate binding site of the rPA-ILNprotein in dictating and modulating its carbohydrate binding properties.This was achieved through extensive site directed mutagenesis tointroduce specific amino acid substitutions in place of these residuesand subsequent evaluation of the carbohydrate binding specificity andaffinity of each of the resulting proteins. In doing so, we also aimedto identify specific amino acid substitutions that promoted specificallyenhanced carbohydrate binding activities.

The role of His50 in defining the α-galactophilic selectivity of thePA-IL protein—The PA-IL protein has been shown to be α-galactophilicwith a preference for glycans displaying α1,4-linked terminal galactose(23). X-ray crystal structures of the protein have been obtained withbound D-galactose and α-galactophilic ligands (23,29). In all of thestructures obtained to date, the terminal galactose is bound in the sameorientation and this is likely due to the large number of interactionsbetween it, the coordinated calcium and specific amino acid side chainsin the binding pocket (FIG. 1B). The PA-IL protein does not bindsignificantly to glycans with terminal β-linked galactose andconsequently no crystal structures with such ligands have been obtained(23). If in silico structural models are generated by overlaying lactoseinto the carbohydrate binding pocket, placing the terminal β1,4-linkedgalactose in the orientation observed in crystal structures obtained todate, it can be seen that the second sugar moiety in the oligosaccharidechain would potentially sterically clash with the His50 residue (FIG.17B). This implies that the His50 residue is likely to be the criticaldeterminant defining the selectivity of the PA-IL protein for glycanswith terminal α-linked galactose by sterically inhibiting binding ofglycans with terminal β1,4-linked galactose.

The impact of His50 substitutions on the carbohydrate bindingspecificity and affinity of rPA-ILN—Our earlier work herein hadindicated that substitution of the His50 residue was particularlycritical in generating lectins capable of binding with high affinity toglycans displaying LacNAc and terminal β1,4-linked galactose. In ExampleB, we assessed the role of this residue in dictating carbohydratebinding properties by introducing 13 independent specific amino acidsubstitutions in its place. Initial qualitative screens of theserPA-ILNm proteins verified that substitution of this residue alone couldsignificantly alter the carbohydrate binding specificity and affinity ofthe protein. Our results also demonstrated that observed changes incarbohydrate binding activities were not simply due to the alleviationof steric restraints imposed by the His50 residue in the carbohydratebinding site as they were dependent on the His50 substitutions made.Some amino acid substitutions simply had a deleterious impact on theoverall carbohydrate binding activity of proteins. However, a number ofspecific amino acid substitutions generated proteins capable of bindingwith high affinity to glycans with terminal β1,4-linked galactose. Amongthese were proteins carrying H50N and H50V substitutions which had alsobeen observed in rPA-ILNm proteins we generated through randommutagenesis in our earlier study herein. Also of particular interest wasthe H50Q protein, which exhibited a dual specificity binding to bothBSA-αGal and BSA-LacNAc glycoconjugates. Through the generation of insilico structural models of these proteins, we explored the potentialstructural basis for the observed carbohydrate binding specificities ofthese proteins.

The carbohydrate binding properties of the H50N protein—The H50N proteinexhibited the highest relative affinity for the BSA-LacNAcglycoconjugate of all of the His50 substitutions made (FIG. 14A) and wasalso the only His50 substituted protein to bind significantly to AsT(FIG. 13). Examination of a predictive structural model of the PA-ILcarbohydrate binding site with a H50N substitution and bound lactosesuggests that such a substitution would not only eliminate stericrestraints (FIG. 17C), that prevent lactose accessing the wild typePA-IL binding site, but that the asparagine side chain could alsopotentially participate in forming a number of productive interactionswith the bound lactose (FIG. 18A). The hydrophilic side chain of theasparagine could potentially contribute one hydrogen bond with theterminal β1,4-linked galactose moiety, thereby compensating for the lossof at least one of the two hydrogen bonds that would otherwise becontributed by histidine with terminal galactose moieties in the wildtype PA-IL. However, it could also contribute two additional hydrogenbonds with the second glucose residue (FIG. 18A). It can also be seenthat the Tyr36 side chain could also engage in the formation of hydrogenbonds with the second glucose residue of lactose. The multipleproductive interactions between the Asn50 and Tyr36 side chains with theglucose moiety of lactose might explain the relatively high affinitythat the H50N protein displays for glycans with LacNAc that we observedin ELLA's. The H50N protein also bound to the BSA-αGal glycoconjugatealthough with significantly lower relative affinity than that observedagainst BSA-LacNAc (FIGS. 13 & 14). Examination of the crystal structureof the wild type PA-IL binding site with the iGb3(Gal-α1,3-Gal-β1,4-Glc) oligosaccharide in the binding site shows thatthe His50 residue contributes two hydrogen bonds with the terminalα1,3-linked galactose and one with the penultimate galactose residue(FIG. 1B). In a model in which the His50 residue is substituted byasparagine, these productive interactions are lost and there ispotentially only one productive interaction between the asparagine sidechain and the terminal galactose (FIG. 19A). This might thereforeaccount for the significant reduction in the relative affinity of theH50N protein for BSA-αGal compared to that of the rPA-ILN proteinobserved in ELLA's.

The carbohydrate binding properties of the H50V protein—The H50V proteinwas also observed to bind strongly to BSA-LacNAc albeit not as stronglyas H50N (FIGS. 13 and 14A). Examination of in silico structural modelsof the PA-IL binding site with a H50V substitution shows that thissubstitution would make the binding site accessible to lactose (FIG.17D). However, unlike the asparagine side chain, the smaller hydrophobicside chain of valine would not be able to contribute to the formation ofhydrogen bonds with lactose (FIG. 18B). This would explain the lowerrelative affinity of the H50V protein for BSA-LacNAc than the H50Nprotein (FIG. 14A) and consequently why, unlike the H50N protein, itfailed to bind significantly to AsT (FIG. 13). The valine side chaincould however contribute to the stabilization of lactose binding throughthe formation of hydrophobic interactions with the second glucoseresidue although these interactions would be likely to be weaker thanthe hydrogen bonds formed by the asparagine side chain in H50N. Asobserved in the H50N model, binding of lactose could also be furtherstabilized by interactions between the side chain of Tyr36 and theglucose residue. While binding of the H50V protein to the BSA-αGalglycoconjugate could be detected, it was very weak. A H50V substitutionwould result in the loss of the productive interactions contributed bythe His50 residue in the PA-IL protein and these would not becompensated for by the replacement valine residue (FIG. 19B). This wouldalso explain why the affinity of H50V for the BSA-αGal was lower thanthat of H50N where the asparagine side chain could at least contributeto binding through the formation of a hydrogen bond with the terminalc-linked galactose.

The carbohydrate binding properties of the HSOQ protein—The H50Q proteindisplayed strong binding to the BSA-LacNAc but it had a significantlylower relative affinity for this glycoconjugate than either the H50N orH50V proteins (FIG. 14A). In structural models of the H50Q binding site,the H50Q substitution would again alleviate the steric constraintsimposed by the His50 residue (FIG. 17E). In FIG. 18C, the glutamine sidechain is depicted in an orientation where it could contribute twohydrogen bonds with the terminal galactose residue of lactose therebycompensating for those normally contributed by His50 in wild type PA-IL.However, in this orientation it would not engage in productiveinteractions with the glucose residue. Such interactions with theglucose residue in the lactose disaccharide, observed in models of boththe H50N and H50V binding site, may again play a greater role instabilizing the binding of lactose than the formation of additionalhydrogen bonds with a terminal galactose residue already well anchoredthrough extensive interactions with amino acid side chains and thecalcium ion. While the Tyr36 side chain could again engage in theformation of positive interactions with the glucose moiety, theinability of the glutamine side chain itself to engage in suchinteractions might account for the higher relative affinity displayed byboth H50N, and H50V, for BSA-LacNAc in ELLA's when compared to that ofH50Q. The glutamine side chain is larger than that of asparagine, andsignificantly larger than that of the hydrophobic valine, and couldadopt a number of orientations other than that depicted in FIG. 18C. Insilico analysis of alternative models of the H50Q binding site suggestedthat in some orientations the glutamine side chain may not be able tocontribute any productive interactions with the lactose moiety and thiscould therefore also explain its lower relative affinity for BSA-LacNAc(data not shown). Some of the potential side chain orientations could infact sterically block lactose from accessing the binding site but, sincethe H50Q protein was observed to bind the BSA-LacNAc glycoconjugate inELLA's, these orientations were discounted.

The H50Q protein exhibited stronger binding to BSA-αGal in ELLA's thanany of the other proteins with single His50 substitutions (FIG. 13), butits affinity for this glycoconjugate was still significantly weaker thanthat of the parental rPA-ILN (FIG. 14B). In a model of the H50Q bindingsite with bound iGb3, it can be seen that the glutamine side chain couldpotentially contribute to the formation of a hydrogen bond with thesecond galactose residue in the oligosaccharide (FIG. 19C). This is incontrast to the H50V model where the side chain of valine might not becapable of contributing any productive interactions and this wouldexplain the higher relative affinity of H50Q for BSA-αGal in ELLA'scompared to that of H50V. In the H50N model, the asparagine side chaincould contribute to binding through formation of a hydrogen bond withthe iGb3 oligosaccharide but this would be formed with the terminal α1-3linked galactose. Again it may therefore be the case that interactionswith the second sugar moiety, or subsequent sugars residues in anoligosaccharide chain, have a greater stabilizing effect onoligosaccharide binding than the formation of additional interactionswith the terminal galactose. This would account for the higher affinityexhibited by the H50Q protein for BSA-αGal compared to that of the H50Nprotein (FIG. 14B).

The role of Gln53 and Asp52 in modulating carbohydrate bindingactivities—Characterisation of the H50N, H50V and H50T proteins, andcomparison with the carbohydrate binding activities of rPA-ILNmE6 (andrPA-ILNmC5), rPA-ILNmF3 and rPA-ILNmB4 respectively, clearly indicatedthat that additional Asp52 and Gln53 substitutions play a role infurther modulating binding carbohydrate binding specificities andaffinities. The Asp52 residue does not participate in forming productiveinteractions with bound iGb3 in the wild type PA-IL binding site (FIG.1B) and so attention was first focused on the Gln53 residue. The H50Nprotein was used as a parental molecule into which 12 specific Gln53substitutions were introduced. Characterisation of the resultingrPA-ILNm revealed only subtle differences in their binding propertiesand most simply exhibited weaker overall binding against the BSAglycoconjugates and AsT. Of particular interest were the resultsobtained for the H50N:Q53G protein. This protein exhibited slightlylower binding signals against AsT compared to H50N and slightly lowerbinding to the BSA-αGal glycoconjugate. This might be expected since aQ53G substitution would result in the loss of the productiveinteractions between the glutamine side chain and either bound lactose(FIG. 18D) or iGb3 (FIG. 19D). Despite this, the rPA-ILNmE6 exhibitssignificantly stronger binding to AsT than either the H50N or H50N:Q53Gproteins, generating signals approximately three fold greater,indicative of a higher affinity for glycoproteins displaying terminalβ1,4-linked galactose. The rPA-ILNmE6 also exhibited greater selectivityfor β-linked galactose over α-linked galactose generating signals nearlytwo fold lower than those observed for the H50N:Q53G protein. As theH50N:Q53G and rPA-ILNmE6 proteins only differ by a single D52Nsubstitution, this additional substitution must be responsible for theenhanced affinity of the rPA-ILNmE6 protein for LacNAc and its greaterselectivity. As the side chain of the substituted Asn52 resent in therPA-ILNmE6 protein would not be expected to interact directly with thebound carbohydrate moiety, it is likely that the D52N substitutionexerts its effect by inducing conformational changes in the carbohydratebinding site or by impacting on the organisation of water moleculeswithin the binding pocket. Structural changes may result in are-orientation of the Asn50 residue so that it interacts more favourablywith glycans with LacNAc. In this respect, the substitution couldpotentially be synergistic with the Q53G substitution as theincorporation of a glycine residue would increase structural flexibilityin the binding site.

Comparison of the binding properties of the H50V and H50T proteins withthose of the rPA-ILNmF3 and rPA-ILNmB4 respectively implied that a Q53Rsubstitution could promote binding to glycans with terminal α-linkedgalactose. To explore this, we introduced a Q53R substitution into theH50V protein to generate a H50V:Q53R double mutant that therefore onlydiffered from the rPA-ILNmF3 protein by a single D52C substitution. ELLAanalysis demonstrated that the resulting H50V:Q53R double mutant didbind to BSA-αGal and that its affinity for this glycoconjugate wascomparable to that of the rPA-ILF3 protein (FIG. 16B). Interestingly thedouble mutant also displayed enhanced affinity for terminal β1,4-linkedgalactose compared to the parental H50V protein. It bound to AsTgenerating responses slightly greater than those of the rPA-ILNmF3 andactually generated significantly higher signals on BSA-LacNAc thaneither the H50V or rPA-ILNmF3 proteins. We subsequently introduced aQ53R substitution into the H50Q protein and the resulting H50Q:Q53Rdouble mutant also displayed an enhanced affinity for the BSA-αGalglycoconjugate (FIG. 16B). However, more surprising was the fact thatH50Q:Q53R double mutant did not bind to the BSA-LacNAc glycoconjugate(FIG. 16A). This clearly indicates that the impact of Gln53substitutions on carbohydrate binding properties is dependent on theamino acid substitution at the His50 position. As none of the mutants wehad constructed exhibited stronger binding to the BSA-αGalglycoconjugate than the parental rPA-ILN protein, we decided tointroduce independent Q53R and Q53E substitutions into the rPA-ILNprotein. As predicted, the resulting Q53R protein displayed an enhancedaffinity for the BSA-αGal glycoconjugate and the Q53E protein was foundto display a slightly higher relative affinity (FIG. 16B).

Final Conclusions—This work successfully demonstrated the critical rolethat the His50 residue plays in dictating the specificity of the PA-ILprotein. We clearly demonstrated that substitution of this residue alonewas sufficient to significantly alter the carbohydrate bindingproperties of the protein. The observation that only specific amino acidsubstitutions promoted high affinity binding to glycans with LacNAc, andterminal β1,4-linked galactose, demonstrated that this was not simplydue to alleviation of steric restraints that might be imposed by theHis50 residue in the carbohydrate binding site of the protein. Throughthe use of structural models generated in silico, we were able toexplore the potential structural basis for the carbohydrate bindingspecificities and affinities displayed by a number of rPA-ILNm proteins.We also demonstrated that both Gln53 and Asp53 substitutions playedsignificant roles in further modulating the binding specificities andaffinities of proteins. Predictive structural models could not explainthe differences in the carbohydrate binding properties of the rPA-ILNmE6protein compared to those of the H50N and H50N:Q53G proteins. These maybe due to conformational changes in structure of the carbohydrate siteinduced by substitution of the Asn52 and Gln53 residues that could notbe predicted and so verification of this will require future solving ofthe structure of these proteins. However, it is also clear from theresults obtained that the final carbohydrate binding properties ofrPA-ILNm proteins is the result of the combined effects of substitutionsat His50, Asn52 and Gln53.

Many of the novel lectins generated in this study will be of use forglycoanalytical applications. While proteins like rPA-ILNmE6 would be ofuse for the detection of terminal β1,4 linked galactose, and LacNAc,others like the H50E protein could provide further biologically relevantinformation about a sample as binding is potentially dependant on thedensity and spatial distribution of glycans. The H50Q, with its dualspecificity for terminal α-linked or β-linked galactose, could be usedfor general detection of terminal galactose while the Q53R and Q53Eproteins, which display enhanced affinity for terminal α-linkedgalactose could be used to detect the presence of this potentiallyimmunogenic sugar moiety. Inclusion of these novel recombinantprokaryotic lectins (RPL's) into any of the currently evolvingglycoanalytical platforms, such as lectin microarrays, wouldsignificantly expand the utility of these platforms. If immobilized tosolid support matrices, these RPL's may also facilitate enhancedglycoselective separations and the purification of glycoproteins andbiotherapeutic molecules.

The invention is not limited to the embodiments described herein but canbe amended or modified without departing from the scope of the presentinvention.

REFERENCES

-   1. Dwek, R. A. (1996). Glycobiology:toward understanding the    function of sugars. Chemical Reviews. 96, 683-720-   2. Drickamer, K., and Taylor, M. E. (2006) Introduction To    Glycobiology, 2nd Edn Ed., Oxford University Press-   3. Katrlik, J., {hacek over (S)}vitel, J., Gemeiner, P., Ko{hacek    over (z)}ár, T., and Tkac, J. (2010). Glycan and lectin microarrays    for glycomics and medicinal applications. Medicinal Research    Reviews. 30, 394-418-   4. Mislovi{hacek over (c)}ová, D., Gemeiner, P., Kozarova, A., and    Ko{hacek over (z)}ár, T. (2009). Lectinomics I. Relevance of    exogenous plant lectins in biomedical diagnostics. Biologia. 64,    1-19-   5. Gemeiner, P., Mislovicova, D., Tkác, J., Svitel, J., P{umlaut    over (ä)}toprstý, V., Hrabárová, E., Kogan, G., and Kozár, T.    (2009). Lectinomics: II. A highway to biomedical/clinical    diagnostics. Biotechnology Advances. 27, 1-15-   6. Ohtsubo, K., and Marth, J. D. (2006). Glycosylation in cellular    mechanisms of health and disease. Cell. 126, 855-867-   7. Chen, S., Zheng, T., Shortreed, M. R., Alexander, C., and    Smith, L. M. (2007). Analysis of cell surface carbohydrate    expression patterns in normal and tumorigenic human breast cell    lines using lectin arrays. Analytical Chemistry. 79, 5698-5702-   8. Dwek, M. V., Lacey, H. A., and Leathem, A. J. C. (1998). Breast    cancer progression is associated with a reduction in the diversity    of sialylated and neutral oligosaccharides. Clinica Chimica Acta.    271, 191-202-   9. Zhao, J., Patwa, T. H., Qiu, W., Shedden, K., Hinderer, R.,    Misek, D. E., Anderson, M. A., Simeone, D. M., and Lubman, D. M.    (2007). Glycoprotein microarrays with multi-lectin detection: unique    lectin binding patterns as a tool for classifying normal, chronic    pancreatitis and pancreatic cancer sera. Journal of Proteome    Research. 6, 1864-1874-   10. Dwek, M. V., Jenks, A., and Leathem, A. J. C. (2010). A    sensitive assay to measure biomarker glycosylation demonstrates    increased fucosylation of prostate specific antigen (PSA) in    patients with prostate cancer compared with benign prostatic    hyperplasia. Clinica Chimica Acta. 411, 1935-1939-   11. Jefferis, R. (2009). Glycosylation as a strategy to improve    antibody-based therapeutics. Nat Rev Drug Discov. 8, 226-234-   12. Raju, T. S. (2008). Terminal sugars of Fc glycans influence    antibody effector functions of IgGs. Current Opinion in Immunology.    20, 471-478-   13. Burton, D. R., and Dwek, R. A. (2006). Sugar determines antibody    activity. Science. 313, 627-628-   14. Marth, J. D., and Grewal, P. K. (2008). Mammalian glycosylation    in immunity. Nat Rev Immunol. 8, 874-887-   15. Stancombe, P. R., Alexander, F. C. G., Ling, R., Matheson, M.    A., Shone, C. C., and Chaddock, J. A. (2003). Isolation of the gene    and large-scale expression and purification of recombinant Erythrina    cristagalli lectin. Protein Expression and Purification. 30, 283-292-   16. Oliveira, C., Teixeira, J. A., and Domingues, L. (2012).    Recombinant lectins: an array of tailor-made glycan-interaction    biosynthetic tools. Critical Reviews in Biotechnology. 0, 1-15-   17. Imberty, A., Mitchell, E. P., and Wimmerova, M. (2005).    Structural basis of high-affinity glycan recognition by bacterial    and fungal lectins. Carbohydrates and glycoconjugates/Biophysical    methods. 15, 525-534-   18. Hu, D., Tateno, H., Kuno, A., Yabe, R., and Hirabayashi, J.    (2012). Directed evolution of lectins with sugar-binding specificity    for 6-sulfo-galactose. Journal of Biological Chemistry. 287,    20313-20320-   19. Yabe, R., Suzuki, R., Kuno, A., Fujimoto, Z., Jigami, Y., and    Hirabayashi, J. (2007). Tailoring a novel sialic acid-binding lectin    from a ricin-B chain-like galactose-binding protein by natural    evolution-mimicry. J Biochem. 141, 389-399-   20. Romano, P. R., Mackay, A., Vong, M., deSa, J., Lamontagne, A.,    Comunale, M. A., Hafner, J., Block, T., Lec, R., and Mehta, A.    (2011). Development of recombinant Aleuria aurantia lectins with    altered binding specificities to fucosylated glycans. Biochemical    and Biophysical Research Communications. 414, 84-89-   21. Gilboa-Garber, N., and Ginsburg, V. (1982) Pseudomonas    aeruginosa lectins. In. Methods in Enzymology, Academic Press-   22. Imberty, A., Wimmerova, M., Mitchell, E. P., and    Gilboa-Garber, N. (2004). Structures of the lectins from Pseudomonas    aeruginosa: insights into the molecular basis for host glycan    recognition. Microbes and Infection. 6, 221-228-   23. Blanchard, B., Nurisso, A., Hollville, E., Tétaud, C., Wiels,    J., Pokorná, M., Wimmerová, M., Varrot, A., and Imberty, A. (2008).    Structural basis of the preferential binding for globo-series    glycosphingolipids displayed by Pseudomonas aeruginosa Lectin I.    Journal of Molecular Biology. 383, 837-853-   24. Beck, A., Wagner-Rousset, E., Bussat, M.-C., Lokteff, M., and    Klinguer-Hamour. (2008). Trends in glycosylation, glycoanalysis and    glycoengineering of therapeutic antibodies and Fc-fusion proteins.    Current Pharmaceutical Biotechnology. 9, 482-501-   25. Arnold, K., Bordoli, L., Kopp, J., and Schwede, T. (2006). The    SWISS-MODEL workspace: a web-based environment for protein structure    homology modelling. Bioinformatics. 22, 195-201-   26. McNicholas, S., Potterton, E., Wilson, K. S., and    Noble, M. E. M. (2011). Presenting your structures: the CCP4mg    molecular-graphics software. Acta Crystallographica Section D. 67,    386-394-   27. Kirkeby, S., and Moe, D. (2002). Lectin interactions with    α-galactosylated xenoantigens. Xenotransplantation. 9, 260-267-   28. Chen, C. P., Song, S. C., Gilboa-Garber, N., Chang, K. S., and    Wu, A. M. (1998). Studies on the binding site of the    galactose-specific agglutinin PA-IL from Pseudomonas aeruginosa.    Glycobiology. 8, 7-16-   29. Cioci, G., Mitchell, E. P., Gautier, C., Wimmerova, M.,    Sudakevitz, D., Perez, S., Gilboa-Garber, N., and Imberty, A.    (2003). Structural basis of calcium and galactose recognition by the    lectin PA-IL of Pseudomonas aeruginosa. FEBS Letters. 555, 297-301-   30. Litterer, L., and Schagat, T. (2007). Protein expression in less    time: a short induction protocol for KRX. Promega Notes. 96, 20-21-   31. Gilboa-Garber, N., and Sudakevitz, D. (1999). The    hemagglutinating activities of Pseudomonas aeruginosa lectins PA-IL    and PA-III_exhibit opposite temperature profiles due to different    receptor types. FEMS Immunol Med Microbiol. 25, 365-369-   32. Hearty, S., Leonard, P., Quinn, J., and O'Kennedy, R. (2006).    Production, characterisation and potential application of a novel    monoclonal antibody for rapid identification of virulent Listeria    monocytogenes. Journal of Microbiological Methods. 66, 294-312-   33. Thompson, R., Creavin, A., O'Connell, M., O'Connor, B., and    Clarke, P. (2011). Optimization of the enzyme-linked lectin assay    for enhanced glycoprotein and glycoconjugate analysis. Analytical    Biochemistry. 413, 114-122-   34. Gilboa-Garber, N., Mizrahi, L., and Garber, N. (1972).    Purification of the galactose-binding hemagglutinin of Pseudomonas    aeruginosa by affinity column chromatography using sepharose. FEBS    Letters. 28, 93-95-   35. Wu, A. M., Song, S. C., Wu, J. H., and Kabat, E. A. (1995).    Affinity of Bandeiraea (Griffonia) simplicifolia Lectin-I,    Isolectin-B4 (BSI-B4) for Gala1-4Gal Ligand. Biochemical and    Biophysical Research Communications. 216, 814-820-   36. Iskratsch, T., Braun, A., Paschinger, K., and Wilson, I. B. H.    (2009). Specificity analysis of lectins and antibodies using    remodeled glycoproteins. Analytical Biochemistry. 386, 133-146-   37. Trimble, R. B., and Atkinson, P. H. (1992). Structural    heterogeneity in the Man₈₋₁₃GIcNAc oligosaccharides from log-phase    Saccharomyces yeast: a one- and two-dimensional 1H NMR spectroscopic    study. Glycobiology. 2, 57-75-   38. Wu, A., Wu, J., Tsai, M.-S., Yang, Z., Sharon, N., and Herp, A.    (2007). Differential affinities of Erythrina cristagalli lectin    (ECL) toward monosaccharides and polyvalent mammalian structural    units. Glycoconjugate Journal. 24, 591-604-   39. Wu, A., Lisowska, E., Duk, M., and Yang, Z. (2008). Lectins as    tools in glycoconjugate research. Glycoconjugate Journal. 26,    899-913-   40. Shields, R. L., Lai, J., Keck, R., O'Connell, L. Y., Hong, K.,    Meng, Y. G., Weikert, S. H. A., and Presta, L. G. (2002). Lack of    fucose on human IgG1 N-linked oligosaccharide improves binding to    human FcγRIII and antibody-dependent cellular toxicity. Journal of    Biological Chemistry. 277, 26733-26740-   41. Qiu, R., and Regnier, F. E. (2005). Use of multidimensional    lectin affinity chromatography in differential glycoproteomics.    Anal. Chem. 77, 2802-2809-   42. Geyer, H., and Geyer, R. (2006). Strategies for analysis of    glycoprotein glycosylation. Biochimica et Biophysica Acta    (BBA)—Proteins & Proteomics. 1764, 1853-1869-   43. Yang, Z., and Hancock, W. S. (2005). Monitoring glycosylation    pattern changes of glycoproteins using multi-lectin affinity    chromatography. Journal of Chromatography A. 1070, 57-64-   44. Walsh, G., and Jefferis, R. (2006). Post-translational    modifications in the context of therapeutic proteins. Nat Biotech.    24, 1241-1252-   45. Walsh, G. (2006). Biopharmaceutical benchmarks 2006. Nat    Biotech. 24, 769-776-   46. Sinclair, A. M., and Elliott, S. (2005). Glycoengineering: The    effect of glycosylation on the properties of therapeutic proteins.    Journal of Pharmaceutical Sciences. 94, 1626-1635-   47. Werner, R. G., Kopp, K., and Schlueter, M. (2007). Glycosylation    of therapeutic proteins in different production systems. Acta    Pdiatrica. 96, 17-22-   48. Kim, H. J., Lee, S. J., and Kim, H.-J. (2008). Antibody-based    enzyme-linked lectin assay (ABELLA) for the sialylated recombinant    human erythropoietin present in culture supernatant. Journal of    Pharmaceutical and Biomedical Analysis. 48, 716-721-   49. Kobata, A. (2000). A journey to the world of glycobiology.    Glycoconjugate Journal. 17, 443-   50. Xu, W., Chen, J., Yamasaki, G., Murphy, J., and Mei, B. (2010).    Lectin Binding Assays for In-Process Monitoring of Sialylation in    Protein Production. Molecular Biotechnology. 45, 248-256-   51. Scallon, B. J., Tam, S. H., McCarthy, S. G., Cai, A. N., and    Raju, T. S. (2007). Higher levels of sialylated Fc glycans in    immunoglobulin G molecules can adversely impact functionality.    Molecular Immunology. 44, 1524-1534

1. A peptide analogue of PA-IL of SEQ ID NO: 1, wherein the peptideanalogue has altered carbohydrate binding specificity, and wherein thepeptide analogue comprises an amino acid substitution at one, two orthree of positions 50, 52 and 53, wherein the amino acid substitution atposition 50 is selected from the group consisting of Ala, Val, Leu, Ile,Met, Phe, Trp, Pro, Ser, Thr, Cys, Tyr, Gly, Asn, Asp, Gln, Glu, Lys,and Arg; wherein the amino acid substitution at position 52 is selectedfrom the group consisting of Ser, Thr, Cys, Asn, Gln, Asp, Glu, Lys, Argand His; wherein the amino acid substitution at position 53 is selectedfrom the group consisting of Ala, Val, Leu, Ile, Met, Phe, Trp, Pro,Gly, Ser, Thr, Cys, Tyr, Asn, Asp, Glu, Lys, Arg and His.
 2. The peptideanalogue of claim 1, wherein the peptide analogue has improved bindingto a carbohydrate having a terminal β galactose over PA-IL of SEQ ID NO:1 and wherein the peptide analogue comprises an amino acid substitutionat position 50 selected from Ala, Val, Leu, Ile, Met, Pro, Ser, Thr,Cys, Asn, Gln, Glu, Lys and Arg.
 3. The peptide analogue of claim 2,wherein the peptide analogue comprises Asn at position 50 and an aminoacid substitution at position 53 selected from the group consisting ofAla, Val, Leu, Ile, Met, Gly, Ser, Thr, Asn, Asp, Glu, Lys, Arg and His.4. The peptide analogue of claim 2, wherein the peptide analoguecomprises Asn at position 50 and Gly at position
 53. 5. The peptideanalogue of claim 2, wherein the peptide analogue comprises Val atposition 50 and an amino acid substitution at position 53 is selectedfrom the group consisting of Ala, Val, Leu, Ile, Met, Gly, Ser, Thr,Asn, Asp, Glu, Lys, Arg and His.
 6. The peptide analogue of claim 1,wherein the peptide analogue has altered carbohydrate bindingspecificity for a carbohydrate having a terminal α-linked galactose overPA-IL of SEQ ID NO: 1 and wherein the peptide analogue comprises anamino acid substitution at position 50 selected from Ala, Val, Leu, Ile,Met, Ser, Thr, Cys, Asn, Gln, Glu, Lys and Arg.
 7. The peptide analogueof claim 6, wherein the peptide analogue comprises Asn at position 50and an amino acid substitution at position 53 selected from the groupconsisting of Ala, Val, Leu, Ile, Met, Gly, Ser, Thr, Cys, Asn, Asp,Glu, Arg, Lys and His.
 8. The peptide analogue of claim 1, wherein thepeptide analogue has enhanced carbohydrate binding specificity for acarbohydrate having a terminal α-linked galactose over PA-IL of SEQ IDNO: 1 and wherein the amino acid substitution at position 53 is selectedfrom the group consisting of Asn, Asp, Glu, Arg and His.
 9. The peptideanalogue of claim 1, wherein the peptide analogue has alteredcarbohydrate binding specificity for a carbohydrate having a terminalα-linked galactose over PA-IL of SEQ ID NO: 1 and wherein the peptideanalogue comprises Val at position 50 and an amino acid substitution atposition 53 selected from the group consisting of Ala, Val, Leu, Ile,Met, Phe, Trp, Pro, Gly, Ser, Thr, Cys, Tyr, Asn, Asp, Glu, Lys, Arg andHis.
 10. The peptide analogue of claim 1, wherein the peptide analoguehas altered carbohydrate binding specificity for a carbohydrate having aterminal α-linked galactose over PA-IL of SEQ ID NO: 1 and wherein thepeptide analogue comprises Gln at position 50 and an amino acidsubstitution at position 53 selected from the group consisting of Ala,Val, Leu, Ile, Met, Phe, Trp, Pro, Gly, Ser, Thr, Cys, Tyr, Asn, Asp,Glu, Lys, Arg and His.
 11. The peptide analogue of claim 1, wherein thepeptide analogue has enhanced carbohydrate binding specificity for acarbohydrate having a terminal β- or a α-linked galactose over PA-IL ofSEQ ID NO: 1 and wherein the peptide analogue comprises an amino acidsubstitution at position 50 selected from Asn, Leu and Gln.
 12. Thepeptide analogue of claim 1, wherein the peptide analogue comprises anamino acid substitution at position 50 selected from Ala, Val, Leu, Phe,Pro, Gly, Ser, Thr, Asn, Gln, Asp, Glu, Lys, and Arg.
 13. The peptideanalogue of claim 1, wherein the peptide analogue comprises Asn atposition 50 and an amino acid substitution at position 53 selected fromthe group consisting of Ala, Val, Leu, Gly, Ser, Tyr, Asn, Asp, Glu,Lys, Arg and His.
 14. The peptide analogue of claim 1, wherein thepeptide analogue comprises an amino acid substitution at position 53selected from Glu, Lys and Arg.
 15. The peptide analogue of claim 1,wherein the peptide analogue comprises Val at position 50 and an aminoacid substitution at position 53 selected from the group consisting ofAla, Val, Leu, Gly, Ser, Tyr, Asn, Asp, Glu, Lys, Arg and His.
 16. Thepeptide analogue of claim 1, wherein the peptide analogue comprises anamino acid substitution at position 52 selected from the groupconsisting His, Asn, Cys, Thr and Arg.
 17. A method for detectingchanges in the glycosylation of a carbohydrate that is optionallyselected from the group consisting of glycoprotein, glycoconjugate andcell surface, the method comprising qualitatively or quantitativelyassessing terminal galactosylation using a peptide analogue of claim 1.18. A method of separating and isolating/purifying biomolecules/cellscomprising a glycoprotein or a glycoconjugate, the method comprisingcontacting the peptide analogue of claim 1 with a solution or suspensioncontaining biomolecules/cells; and separating any biomolecules/cells notbound by the peptide analogue.
 19. A method for detecting changes in theglycosylation of a carbohydrate, the method comprising qualitatively orquantitatively assessing terminal galactosylation using a peptideanalogue of claim
 2. 20. A method of separating and isolating/purifyingbiomolecules/cells comprising a glycoprotein or a glycoconjugate, themethod comprising contacting the peptide analogue of claim 2 with asolution or suspension containing biomolecules/cells; and separating anybiomolecules/cells not bound by the peptide analogue.